Tumour suppression with use of human placental perfusate and auxiliary natural killer cells recovered from human placenta

FIELD: medicine.

SUBSTANCE: using the human placental perfusate cells in preparing a therapeutic agent for suppressing tumour cells proliferation in an individual having the tumour cells, wherein the placental perfusate cells represent a collection of nuclear cells of the placental perfusate. Using natural killer cells of CD56+, CD16- recovered from the placenta for preparing the therapeutic agent for suppressing the tumour cells proliferation in the individual having the tumour cells. Using the combined natural killer cells in preparing the therapeutic agent for suppressing the tumour cells proliferation in the individual having the tumour cells, wherein the above combined natural killer cells comprise the natural killer cells recovered from the placental perfusate, and the natural killer cells recovered from the umbilical blood, and wherein the umbilical blood is recovered from the placenta, which is used to prepare the above placental perfusate. A method for suppressing the tumour cells proliferation in vitro, involving the tumour cells contact to the human placental perfusate cells, wherein the placental perfusate cells represent the collection of the nuclear cells from the placental perfusate. The method for suppressing the tumour cells proliferation in vitro, involving the tumour cell contact to the number of the natural killer cells prepared of placental CD56+, CD16-. The method for suppressing the tumour cells proliferation in vitro, involving the tumour cells contact to the combined natural killer cells, wherein the above combined natural killer cells involve the natural killer cells recovered from the placental perfusate, and the natural killer cells recovered from the umbilical blood, and wherein the umbilical blood is recovered from the placenta, which is used for prepare the above placental perfusate. A composition applicable in suppressing the tumour cells proliferation, containing the recovered natural killer cells of CD56+, CD16-, wherein the above natural killer cells are recovered from the placental perfusate, and wherein the above natural killer cells make at least 50% cells in the composition.

EFFECT: placental perfusate cells and methods for using them enable suppressing the tumour cells proliferation effectively.

40 cl, 13 tbl, 6 ex, 11 dwg

 

In this application claims priority on provisional application for U.S. patent No. 60/995763, filed September 28, 2008, and provisional application for U.S. patent No. 61/090555, filed August 21, 2008, the description of which is entirely included in the present invention by reference.

1. The technical field to which the invention relates

In the present description provides methods of inhibiting the growth or proliferation of cancer cells by contacting tumor cells with placental perfusion solution, with cells isolated from placental perfusion solution, with natural killer cells from the placenta, in particular from placental perfusion solution, and/or combined natural killer cells, including natural killer cells from the placenta, in particular from placental perfusion solution, and natural killer cells from umbilical cord blood. In addition, the present invention provides methods of producing a unique population of natural killer cells from the placenta, for example, from placental perfusion solution, in particular from placental perfusion solution man. In addition, the present invention provides methods of using placental perfusion solution and separated from his natural killer cells to suppress the proliferation of cancer cells.

2. The level of technology

Placental perfusion solution soda is separated by the accumulation of cells of the placenta, obtained by passing a solution for perfusion through the vasculature of the placenta and the extraction solution for perfusion of the vascular network from the maternal surface of the placenta or both of them. Methods perfusion of placental mammals can be found, in particular, in U.S. patent No. 7045146 and in U.S. patent No. 7255879. The population of placental cells, isolated using perfusion, heterogeneous and includes hematopoietic (CD34+) cells, nucleated cells, such as granulocytes, monocytes and macrophages, a small percentage (less than 1%) stem cells of the placenta associated with the substrate tissue culture, and natural killer cells. To date no one has described the use of placental perfusion solution or population of placental cells from perfusion solution for suppressing proliferation of cancer cells.

Natural killer cells (NK) are cytotoxic lymphocytes, which are a major component of the innate immune system of the person. NK cells do not Express receptors T-cell antigen (TCR), CD3 or receptor surface immunoglobulin of b-cells, but usually Express people surface markers CD16 (FcγRIII) and CD56. NK cells exert a cytotoxic effect; small granules in their cytoplasm contain special proteins, such as perforin and proteases, known as granzyme. When you is in order his immediate vicinity of the cells, which is scheduled for destruction, perforin forms pores in the cell membrane of target cells, through which can penetrate granzyme and related molecules that induce apoptosis. One of Gerasimov, Grasim (also known as Grasim 2 and cytotoxic T-lymphocyte-associated carinatherese 1), is seanastheysay, which plays a crucial role in the rapid induction of apoptosis of target cells mediated by cells of the immune response.

NK cells are activated in response to interferons or cytokines produced by macrophages. Activated NK cells called activated lymphokines killer cells (LAK). NK cells have two types of surface receptors, designated as “activating receptor” and “inhibiting receptor”, which regulate the cytotoxic activity of the cells.

Among the other which has active substances NK cells play a role in the rejection of tumors by the owner. Since cancer cells have reduced expression or not have the expression of MHC molecules of class I, then they can become targets for NK cells. Collected clinical data suggests that identical haplotype transplantation, NK cells isolated from PBMC or bone marrow mediates powerful antileykemichesky action without showing Zam is based graft-versus-host (GVHD). Cm. Ruggeri et al.,Science295:2097-2100 (2002)). Natural killer cells can be cells that are or have reduced levels of protein major histocompatibility complex (MHC). Activated and multiplied NK cells and LAK cells were used as inex vivotherapy, andin vivothe treatment of patients with cancer at a late stage, while progress has been made in the treatment of diseases associated with bone marrow, such as leukemia, breast cancer, and some types of lymphoma. Treatment using LAK cells requires that the patient first received IL-2, and then passed lycopenes with subsequent ex vivo incubation and rearing of collected autologous blood cells in a nutrient medium for several days in the presence of IL-2. To complete the treatment LAK cells should be subjected reinfused with relatively high doses of IL-2. Specified cleansing treatment is expensive and can cause serious side effects. These include fluid retention, pulmonary edema, falling blood pressure, and high temperature.

Despite the favorable properties of NK cells to kill tumor cells and virus-infected cells, they still find it hard to work and hard to use in immunotherapy, mainly due to the difficulties to maintain the ability of NK cells n telepathise to target and destroy tumor cells in the process of breeding. Thus, in the art a need for an easily accessible source of natural killer cells.

3. The invention

In the present invention proposes the use of placental perfusion solution; cells from placental perfusion solution, in particular, the aggregate nucleated cells from placental perfusion solution; combinations of cells placental perfusion solution and cells in umbilical cord blood; and/or natural killer cells from the placenta, in particular, natural killer cells from placental perfusion solution and natural killer cells, obtained by fermentation of the tissues of the placenta, with the aim of suppressing the proliferation of tumor cells.

In accordance with one aspect, the present invention proposes a method of suppressing the proliferation of tumor cells or populations of tumor cells which comprises contacting the tumor cells or population of tumor cells with placental perfusion solution man. In a specific embodiment of this method the tumor cell is a cancer cell of the blood. In another specific embodiment of the present invention, tumor cells are cancer cells in the blood. In another specific embodiment of the present invention, the tumor cell is a cell is a solid tumor. In another specific options the ante implementation of the present invention, tumor cells are cells of a solid tumor. In another specific embodiment of the present invention, the tumor cell is a primary cell carcinoma of the epithelium of the ducts, the cell leukemia, cell acute T-cell leukemia cell chronic myeloid lymphoma (CML), the cell acute myelogenous leukemia, cell chronic myelogenous leukemia (CML), cell lung carcinoma, cell adenocarcinoma of the colon, cell histiocytoses lymphoma, multiple myeloma cell, a retinoblastoma cell, the cell colorectal carcinoma or colorectal cell adenocarcinoma. In another specific embodiment, the present invention specified communication occurs in conditions ofin vitro. In another specific embodiment, the present invention specified communication occurs in conditions ofin vivo. In a more specific embodiment of the present invention the specifiedin vivocommunication occurs in a person.

In another specific embodiment, the present invention specified placental perfusion solution is a perfusion solution, which is passed through a vasculature of the placenta, in particular, only through the vasculature of the placenta. In another specific embodiment, the present invention specified placental perfusion solution is passed through a vascular network Platz the options and assembled with the maternal surface of the placenta. In another specific embodiment of the present invention, all or almost all (in particular, more than 90%, 95%, 98% or 99%) of the cells in the specified placental perfusion solution are the cells of the fetus. In another specific embodiment, the present invention placental perfusion solution contains cells of fetal and maternal cells. In a more specific embodiment of the present invention the cells of the fetus in the specified placental perfusion solution are less than approximately 90%, 80%, 70%, 60% or 50% of the cells in the specified perfusion solution. In another specific embodiment, the present invention specified perfusion solution get, skipping of 0.9% NaCl solution through the vasculature of the placenta. In another specific embodiment, the present invention specified perfusion solution includes the culture medium. In another specific embodiment, the present invention specified perfusion solution is subjected to treatment to remove many red blood cells.

In accordance with another aspect, the present invention proposes a method of suppressing the proliferation of tumor cells or a variety of tumor cells which comprises contacting the tumor cells or a variety of tumor cells with many cells placental perfusion solution. In another specific embodiment, this is subramania specified set of cells placental perfusion solution represents or includes the totality of nucleated cells from placental perfusion solution. In another specific embodiment, the present invention specified placental perfusion solution or cells placental perfusion solution, in particular the combination of nucleated cells from placental perfusion solution, processed to remove at least one type of cells. In another specific embodiment, the present invention specified communication occurs in conditions ofin vitro. In another specific embodiment, the present invention specified communication occurs in conditions ofin vivo. In a more specific embodiment of the present invention the specifiedin vivocommunication occurs in a mammal, in particular in man. In another specific embodiment of the present invention, these cells are placental perfusion solution was processed in order to enrich at least one type of cells, in particular cells CD56+. In another specific embodiment, the present invention cells CD56+are natural killer cells CD56+CD16‾, in particular supporting natural killer cells of the placenta (PINK), which, for example, isolated cells from placental perfusion solution and the placental cells obtained by mechanical or enzymatic degradation of the tissue of the placenta. In another to the specific embodiment of the present invention mentioned CD56 +cells are selected using CD56-linked beads. In another specific embodiment, the present invention indicated CD56+cells are cells that show significantly lower expression of NKG2D, NKp46 or CD94 than the equivalent number of CD56+CD16+natural killer cells. In another specific embodiment, the present invention PINK cells are CD3‾. In a more specific embodiment of the present invention, at least 50% of the cells in these cells placental perfusion solution are listed CD56+cells. In a more specific embodiment of the present invention, when CD56+cells make up at least 50% of these cells placental perfusion solution, the tumor cell is a primary cell carcinoma of the epithelium of the ducts, the cell leukemia, cell acute T-cell leukemia cell chronic myeloid lymphoma (CML), the cell acute myelogenous leukemia, cell chronic myelogenous leukemia (CML), cell lung carcinoma, cell adenocarcinoma of the colon, the cell histiocytoses lymphoma, multiple myeloma cell, a retinoblastoma cell, the cell colorectal carcinoma or colorectal cell adenocarcinoma. In specific embodiments, the implementation of this is about the invention of the specified communication occurs in conditions of in vitro. In another specific embodiment, the present invention specified contacts is a contactsin vivoin particular in a mammal, such as man.

In accordance with another aspect, the present invention proposes a method of suppressing the proliferation of tumor cells or a variety of tumor cells which comprises contacting the tumor cells or a variety of tumor cells with lots of natural killer cells from the placenta, in particular PINK cells. In a specific embodiment, the present invention natural killer cells from the placenta are natural killer cells derived from placental perfusion solution. In another specific embodiment, the present invention natural killer cells are natural killer cells, which receive physical destruction and/or enzymatic breakdown of the tissues of the placenta. In another specific embodiment, the present invention natural killer cells are natural killer cells CD56+CD16‾, in particular PINK cells. In another specific embodiment of the present invention these natural killer cells is chosen in particular from cells placental perfusion solution or cells derived p is the physical destruction and/or enzymatic degradation of the tissue of the placenta, using CD56-of conjugated beads. In another specific embodiment, the present invention natural killer cells are CD3‾. In a specific embodiment of the present invention the set of natural killer cells is at least 80% of the cells in the cell population, which contains natural killer cells. In another specific embodiment, the present invention specified communication occurs in conditions ofin vitro. In another specific embodiment, the present invention specified contacts in terms ofin vivooccurs in mammals, particularly in man.

In another specific embodiment of the method according to the present invention the specified array of natural killer cells represent cells that show significantly lower expression of NKG2D, NKp46 or CD94 than the equivalent number of CD56+CD16+natural killer cells. In another specific embodiment, the present invention specified variety of natural killer cells, in particular PINK cell expresses one or more molecules of the microRNA hsa-miR-100, hsa-miR-127, hsa-miR-211, hsa-miR-302c, hsa-miR-326, hsa-miR-337, hsa-miR-497, hsa-miR-512-3p, hsa-miR-515-5p, hsa-miR-517b, hsa-miR-517c, hsa-miR-518a, hsa-miR-518e, hsa-miR-519d, hsa-miR-520g, hsa-miR-520h, hsa-miR-564, hsa-miR-566, hsa-miR-618, and/or hsa-miR-99a with children who tiramin higher level, than natural killer cells in peripheral blood.

In another specific embodiment, the present invention specified variety of natural killer cells, in particular PINK cells are in contact with a number of immunomodulatory compounds and in the course of time, which is sufficient for the specified set of natural killer cells could Express detective more granzyme In than the equivalent number of specified natural killer cells that are not contacted with the specified immunomodulatory compound. In a more specific embodiment of the present invention specified immunomodulatory compound is a lenalidomide or pomalidomide. In another specific embodiment, the present invention specified variety of natural killer cells, in particular PINK cells, in contact with a number of immunomodulatory compounds and for a time sufficient to ensure that these natural killer cells could be detektiruya greater cytotoxicity against these tumor cells than an equivalent number of specified natural killer cells that are not contacted with the specified immunomodulatory compound, in particular with lenalidomide or pomalidomide. In others the GOM specific embodiment of the present invention specified variety of natural killer cells, in particular, PINK cells, and expresses one or more of BAX, CCL5, CCR5, CSF2, FAS, GUSB, IL2RA or TNFRSF18 with a higher level than an equivalent number of specified natural killer cells that are not contacted with the specified immunomodulatory compound. In another specific embodiment, the present invention specified variety of natural killer cells, in particular PINK cell expresses one or more of ACTB, BAX, CCL2, CCL3, CCL5, CCR5, CSF1, CSF2, ECE1, FAS, GNLY, GUSB, GZMB, IL1A, IL2RA, IL8, IL10, LTA, PRF1, PTGS2, SKI and TBX21 with a higher level than an equivalent number of specified natural killer cells that are not contacted with the specified immunomodulatory compound.

In another embodiment, the present invention natural killer cells from the placenta combined with natural killer cells from another source, in particular, from the blood of the placenta and/or umbilical cord blood, for example, to obtain the combined natural killer cells. In the present description the expression “natural killer cells from the placenta does not include natural killer cells from umbilical cord blood or the blood of the placenta. In a more specific embodiment of the present invention natural killer cells from the placenta combined with natural killer cells from another source in the ratio will bring the flax 100:1, 95:5, 90:10, 85:15, 80:20, 75:25, 70:30, 65:35, 60:40, 55:45: 50:50, 45:55, 40:60, 35:65, 30:70, 25:75, 20:80, 15:85, 10:90, 5:95, 100:1, 95:1, 90:1, 85:1, 80:1, 75:1, 70:1, 65:1, 60:1, 55:1, 50:1, 45:1, 40:1, 35:1, 30:1, 25:1, 20:1, 15:1, 10:1, 5:1, 1:1, 1:5, 1:10, 1:15, 1:20, 1:25, 1:30, 1:35, 1:40, 1:45, 1:50, 1:55, 1:60, 1:65, 1:70, 1:75, 1:80, 1:85, 1:90, 1:95, 1:100 and so on

In specific embodiments, the implementation of the present invention combined natural killer cells do not grow in a nutrient medium, and they include: detective more natural killer cells CD3‾CD56+CD16‾ than an equivalent number of natural killer cells from peripheral blood; detective fewer natural killer cells CD3‾CD56+CD16+than an equivalent number of natural killer cells from peripheral blood; detective more natural killer cells CD3‾CD56+KIR2DL2/L3+than an equivalent number of natural killer cells from peripheral blood; detective fewer natural killer cells CD3‾CD56+NKp46+than an equivalent number of natural killer cells from peripheral blood; detective more natural killer cells CD3‾CD56+NKp30+than an equivalent number of natural killer cells from peripheral blood; detective more natural killer cells CD3‾CD56+2B4+than an equivalent number of natural killer cells from perifericheskoi blood; or detective more natural killer cells CD3‾CD56+CD94+than an equivalent number of natural killer cells from peripheral blood. In other specific embodiments, the implementation of the present invention combined natural killer cells are cultivated in a nutrient medium, and they include: detective fewer natural killer cells CD3‾CD56+KIR2DL2/L3+than an equivalent number of natural killer cells from peripheral blood; detective more natural killer cells CD3‾CD56+NKp46+than an equivalent number of natural killer cells from peripheral blood; detective more natural killer cells CD3‾CD56+NKp44+than an equivalent number of natural killer cells from peripheral blood; detective more natural killer cells CD3‾CD56+NKp30+than an equivalent number of natural killer cells from peripheral blood.

In a specific embodiment, any of the above methods, the tumor cell is a solid tumor cell. In another specific embodiment of the present invention, the tumor cell is a cell of liquid tumors, in particular, blood cell tumors. In more specific embodiments, the implementation of the crust is asego invention, the tumor cell is a primary cell carcinoma of the epithelium of the ducts, cell leukemia, cell acute T-cell leukemia cell chronic myeloid lymphoma (CML), cell acute myelogenous leukemia, cell chronic myelogenous leukemia (CML) cell lung carcinoma, cell adenocarcinoma of the colon, cell histiocytoses lymphoma, multiple myeloma cell, a retinoblastoma cell, cell colorectal carcinoma or colorectal cell adenocarcinoma.

In accordance with another aspect, the present invention features a composition comprising isolated from placental natural killer cells CD56+, CD16‾, in particular PINK cells. In a specific embodiment of the present invention these natural killer cells of the placenta separated from the placental perfusion solution. In another specific embodiment of the present invention these natural killer cells of the placenta separated from the placenta by physical destruction and/or enzymatic degradation of placental tissue. In another specific embodiment of the present invention these natural killer cells constitute at least 50% of the cells in the composition. In a specific embodiment of the present invention these natural killer cells constitute at least 80% of the cells in the composition. In another specific Varian is the first implementation of the present invention the specified composition includes selected natural killer cells CD56 +, CD16+. In a more specific embodiment of the present invention these natural killer cells CD56+, CD16+obtained from another individual than these natural killer cells CD56+, CD16‾. In another specific embodiment of the present invention these selected natural killer cells CD56+, CD16‾ obtained from one individual. In a more specific embodiment of the present invention these selected natural killer cells CD56+, CD16‾ represent natural killer cells, from at least two different individuals. In another specific embodiment of the present invention these natural killer cells of the placenta, in particular PINK cells multiply.

In a more specific embodiment of the present invention, the composition includes natural killer cells of the placenta and natural killer cells from another source. In a specific embodiment, the present invention specified by another source are cells from umbilical cord blood and/or blood from the umbilical cord. In another specific embodiment, the present invention specified by another source is peripheral blood. In more specific embodiments, the implementation of the present invention naturallyclassy-killers from the placenta combined with natural killer cells from another source in a ratio of about 100:1, 95:5, 90:10, 85:15, 80:20, 75:25, 70:30, 65:35, 60:40, 55:45: 50:50, 45:55, 40:60, 35:65, 30:70, 25:75, 20:80, 15:85, 10:90, 5:95, 100:1, 95:1, 90:1, 85:1, 80:1, 75:1, 70:1, 65:1, 60:1, 55:1, 50:1, 45:1, 40:1, 35:1, 30:1, 25:1, 20:1, 15:1, 10:1, 5:1, 1:1, 1:5, 1:10, 1:15, 1:20, 1:25, 1:30, 1:35, 1:40, 1:45, 1:50, 1:55, 1:60, 1:65, 1:70, 1:75, 1:80, 1:85, 1:90, 1:95, 1:100 and so on

In another specific embodiment, the present invention this composition is a dedicated placental perfusion solution. In a more specific embodiment of the present invention specified placental perfusion solution obtained from the same individual, and that these natural killer cells. In a more specific embodiment of the present invention specified placental perfusion solution is a placental perfusion solution from another individual than these natural killer cells. In another specific embodiment of the present invention, all or almost all (in particular, more than 90%, 95%, 98% or 99%) of the cells in the specified placental perfusion solution are the cells of the fetus. In another specific embodiment, the present invention placental perfusion solution includes cells of fetal and maternal cells. In a more specific embodiment of the present invention the cells of the fetus in the specified placental perfusion solution are less than approximately 90%, 80%, 70%, 60% or 50% of the cells in the specified perfusion solution. In another specific embodiment, the OS is the hope of the present invention, these perfusion solution obtained when the passage of 0.9% NaCl solution through the vasculature of the placenta. In another specific embodiment, the present invention specified perfusion solution includes the culture medium. In another specific embodiment, the present invention specified perfusion solution was processed in order to remove many red blood cells.

In another specific embodiment, the present invention specified composition comprises cells of placental perfusion solution. In a more specific embodiment of the present invention, these cells are placental perfusion solution obtained from the same individual, and that these natural killer cells. In another more specific embodiment of the present invention, these cells are placental perfusion solution obtained from another individual than these natural killer cells. In another specific embodiment, the present invention specified composition includes a dedicated placental perfusion solution and the selected cells placental perfusion solution, with the specified selected placental perfusion solution and these selected cells placental perfusion solution obtained from different individuals. In another more specific embodiment of embodiment of any of the above embodiments of the present invention, including placental perfusion solution specified placental perfusion solution is the FDS is th placental perfusion solution, at least two individuals. In another more specific embodiment of embodiment of any of the above embodiments of the present invention, including cells of placental perfusion solution, these selected cells placental perfusion solution taken from at least two individuals. This composition may further include a dedicated PINK cells, while PINK cells obtained from a different individual than the specified placental perfusion solution or cells perfusion solution.

In accordance with another aspect, the present invention proposes a method of allocation of natural killer cells of the placenta, which includes many of the cells of the placenta and the allocation of natural killer cells from a specified set of cells of the placenta. In a specific embodiment of the present invention the cells of the placenta are or include cells of placental perfusion solution, in particular, the total number of nucleated cells from placental perfusion solution. In another specific embodiment, the present invention specified set of cells of the placenta represents or includes cells of the placenta, obtained by mechanical and/or enzymatic degradation of placental tissue. In another embodiment of the present invention the specified highlighting spend with IP is the use of one or more antibodies. In a more specific embodiment of the present invention specified one or more antibodies represent one or more antibodies to CD3, CD16 or CD56. In a more specific embodiment of the present invention the specified selection includes separation of cells CD56+from cells CD56‾ in a specified set of cells of the placenta. In a more specific embodiment of the present invention the specified selection includes separation of the placental cells CD56+, CD16‾ from placental cells that are CD56‾ or CD16+. In a more specific embodiment of the present invention the specified selection includes separation of the placental cells CD56+, CD16‾, CD3‾ from placental cells that are CD56‾, CD16+or CD3+. In another embodiment, the present invention this method of allocation of natural killer cells of the placenta leads to a population of placental cells that, at least on 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or, at least 99% consists of natural killer cells CD56+, CD16‾.

In some embodiments, the above methods cells placental perfusion solution propagated in culture. In various embodiments, implementation of the present invention, these cells multiply, at least, about, or no more than che is within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 days. In a specific embodiment of the present invention, these cells are placental perfusion solution multiply in the presence of feeder and/or in the presence of at least one cytokine. In a more specific embodiment of the present invention the specified feeder includes cells C or mononuclear cells of peripheral blood. In another more specific embodiment, the present invention is specified, at least one cytokine is an interleukin-2.

3.1. Definitions

In the context of the present description “combined natural killer cells are natural killer cells, in particular, are compatible with each other umbilical cord and placental perfusion solution with placental perfusion solution obtained from the same placenta, and umbilical cord blood. Natural killer cells from both sources emit separately or at the same time and combine.

In this description of “PINK” and “PINK squares” auxiliary natural killer cells of the placenta derived from human placenta, in particular from placental perfusion solution person, or tissues of the placenta, which are subjected to mechanical and/or enzymatic degradation. These cells represent the Wallpaper CD56 +and CD16‾, which, for example, determined by flow cytometry, in particular, determine the method of fluorescently-activated cell sorting using antibodies to CD56 and CD16. PINK cells are not derived from umbilical cord blood or peripheral blood.

In the present description the expression “placental perfusion solution” means a solution for perfusion, which is omitted, at least through a portion of the placenta, such as placenta of man, in particular, through the vasculature of the placenta, including a large number of cells collected solution for perfusion during the passage through the placenta.

In the present description the expression “cells placental perfusion solution” means nucleated cells, in particular all nucleated cells, which are allocated to, or can be isolated from placental perfusion solution.

In the present description, the expression “inhibition of tumor cells, inhibiting proliferation of tumor cells”, etc. includes the slowdown in the growth of tumorous cells, in particular, by destroying one or more tumor cells in the specified population of tumor cells, for example, by contacting a specified population of tumor cells with PINK cells, populations of cells, including PINK cells, combined natural killer cells, populations of cells, including combined is consistent natural killer cells, with placental perfusion solution person, etc.

4. Brief description of drawings

In Fig.1 shows the results of flow cytometry using antibodies against CD3 and antibodies against CD56 cell selected with the help of beads CD56 of placental perfusion solution people (HPP). Most of the isolated cells are CD56+CD3‾.

In Fig.2 shows the production of cytokines PINK cells and/or tumor cells during cultivation in a nutrient medium for 24 h Fig.2A shows the secretion of interferon gamma (IFNγ) auxiliary natural killer cells (PINK), isolated from placental perfusion solution, individually or in the presence of tumor cells KG-1a. PINK cells and cells KG-1a grown in a nutrient medium individually or in combination at a ratio of 1:1. Y axis: picogram IFNγ produced by the cultures. Fig.2B shows the secretion of granulocyte-macrophage colony-stimulating factor (GM-CSF) PINK cells individually or in the presence of tumor cells KG-1a. PINK cells and cells KG-1a grown in a nutrient medium individually or in combination at a ratio of 1:1. Y axis: picogram GM-CSF produced by the cultures.

In Fig.3 shows the cytotoxicity PINK cells towards tumor cells and KG-1a at joint cultivation for 24 h in nutrient medium at a ratio of 1:1,5:1, 10:1 or 20:1 PINK cells to tumor cells. X axis: the ratio of the PINK cells to tumor cells. Y axis: percentage of dead tumor cells compared with tumor cells without PINK cells.

In Fig.4 shows the cytotoxicity of natural killer cells (NK) placenta and natural killer cells in peripheral blood (PB), which is grown in a nutrient medium for 21 days, compared to cells C. The magnitude of the errors represent standard deviation of 4 units grown NK cells of placenta or 3 units grown NK cells in peripheral blood.

In Fig.5 shows the cytotoxicity whole placental perfusion solution (HPP), which, as obtained from the placenta, in relation to tumor cells KG-1a at joint cultivation for 24 h in culture medium in a ratio of 1:1, 5:1, 10:1, 20:1 or 100:1 HPP cells to tumor cells. The X-axis: ratio of HPP cells to tumor cells. Y axis: percentage of dead tumor cells compared with tumor cells without cell HPP.

In Fig.6 shows the cytotoxicity whole placental perfusion solution, which, as obtained from the placenta and umbilical cord blood (UCB), in relation to tumor cells KG-1a at joint cultivation in a nutrient medium within 48 h for a series of dilutions 100:1, 50:1, 25:1, 12,5:1, 6,25:1, 3,12:1, 1,56:1 or to 0.78:1 HPP cells or UCB cells to tumor cells. About the ü X: the ratio of HPP cells or umbilical cells to tumor cells. Y axis: percentage of dead tumor cells after co-cultivation for 48 h in the medium, compared with tumor cells without HPP cells or cells of the umbilical cord.

In Fig.7 shows the cytotoxicity whole placental perfusion solution, which, as obtained from the placenta, in relation to tumor cells KG-1a at joint cultivation in a nutrient medium within 48 h for a series of dilutions 100:1, 50:1, 25:1, 12,5:1, 6,25:1, 3,12:1, 1,56:1 or to 0.78:1 HPP cells to tumor cells. The perfusion solution or used as it is received, or activate for 24 h with 100 units/ml or 1000 units/ml interleukin-2 (IL-2). The X-axis: ratio of HPP cells to tumor cells. Y axis: percentage of dead tumor cells after co-cultivation in a nutrient medium for 48 h, compared with tumor cells without cell HPP.

In Fig.8 shows the cytotoxic effect of placental perfusion solution person in relation to the listed lines of tumor cells after cultivation in a nutrient medium with HPP cells or cells UCB in respect of 50:1 to tumor cells. Fig.8A: culturing in a nutrient medium for 24 h Fig.8B: culturing in a nutrient medium within 48 h of the X Axis: tested cell lines. Y axis: percentage of dead tumor cells after co-cultivation is nutritional in the second environment, compared to the number of tumor cells in the absence of tumor cells.

In Fig.9 shows the production of IFNγ cells HPP, which is grown in a nutrient medium together with the cells KG-1a with different relations of HPP cells to tumor cells. X axis: the conditions of the experiment, including the ratio of HPP cells to tumor cells. Y axis: the levels of IFNγ per milliliter after co-cultivation in a nutrient medium within 24 hours

In Fig.10 shows the production of IFNγ by HPP cells or cells at UCB joint cultivation in a nutrient medium with a number of tumor cells. The HPP cells or cells UCB jointly cultivated in a nutrient medium in the ratio of 50:1 with tumor cells within 24 h (Fig.10A) or 48 h (Fig.10V). The levels of IFNγ determine fluorescence analysis method Luminex (HCYTO-60K-03, Millipore). X axis: tested cell lines. Y axis: picogram IFNγ produced by HPP cells or cells in UCB compared with picogram IFNγ produced in the absence of tumor cells.

Fig.11 shows the decrease in tumor size after injection of 2×107cells placental perfusion solution people (HPP) mice with tumor cells KG-1, the volume of which is approximately 332 mm3. Intratumoral injection of HPP cells injected subcutaneously directly into the tumor. Intravenous - cells HPP impose nutrion is O. Control - the introduction of only one carrier. Tumor volume is specified in mm3.

5. Detailed description of the invention

In the present invention proposes the use of placental perfusion solution, cells placental perfusion solution and/or isolated from the perfusion solution of natural killer cells (“PINK”), which is obtained from the placenta, with the aim of inhibiting the growth or proliferation of cancer cells, and many tumor cells. In particular, the present invention offers natural killer cells (NK) and populations of NK cells isolated from placental perfusion solution, in particular placental perfusion solution person, or isolated from placental tissue, which came under mechanical and/or enzymatic degradation, suggests ways of obtaining NK cells, as well as methods of using these cells. In the present invention are also populations of cells, in particular of a population of placental cells that comprise natural killer cells. Methods of obtaining placental perfusion solution and methods of obtaining cells from placental perfusion solution below in section 5.1. Natural killer cells isolated from placental perfusion solution, and methods of obtaining these cells below in section 5.2. The methods of using placental perfusion solution, cells isolated from placental perfusion solution, or straight is the major killer cells, isolated from placental perfusion solution, in particular an auxiliary natural killer cells, to inhibit proliferation of tumor cells below in section 5.3.

5.1. Placental perfusion solution

5.1.1. Compositions for collecting cells

Proposed in the present invention placental perfusion solution, the cells of placental perfusion solution and natural killer cells isolated from placental perfusion solution can be collected by perfusion of a mammal, in particular, by perfusion post-partum human placenta, using the composition for collecting placental cells. The perfusion solution can be collected from the placenta by perfusion of the placenta in any physiologically acceptable solution, in particular saline, culture medium, or a more sophisticated design to collect cells. Compositions for collecting cells suitable for carrying out perfusion of the placenta and to collect and preserve cells perfusion solution, in particular, the total number of nucleated cells placental perfusion solution, or PINK cells, described in detail in the patent application U.S. No. 2007/0190042, which is entirely included in this description by reference.

Composition for collection of cells can be any physiologically acceptable solution, which is suitable for collection and/or cultivation in a nutrient medium stem glue is OK, for example, a physiological solution (in particular, phosphate buffered saline solution creba, modified solution of creba, the solution of the Needle, a 0.9% NaCl solution and so on), culture medium (in particular, DMEM, H. DMEM and so on), etc.

Composition for collection of cells may include one or more components that tend to keep the cells from the placenta, i.e., capable of preventing cell death of the placenta or delay cell death of the placenta, to reduce the number of placental cells in a population of cells that die, or to have a similar effect, from the moment of collection until the time of cultivation in a nutrient medium. Such components can be, for example, inhibitors of apoptosis (in particular, the caspase inhibitor or inhibitor of JNK); vasodilating agent (in particular, magnesium sulfate, antihypertensive agents, atrial naturethese peptide (ANP), adrenocorticotropin, corticotropin-releasing hormone, sodium nitroprusside, hydralazine, adenosine triphosphate, adenosine, indomethacin, or magnesium sulfate, a phosphodiesterase inhibitor, and so on); an inhibitor of necrosis (in particular, 2-(1H-indol-3-yl)-3-pentylaniline, dithiocarbamate of pyrrolidine or clonazepam); inhibitor of TNF-α; and/or perfluorocarbons, able to carry oxygen (in particular, perforative, perftordecaline and so on).

<> Composition for collection of cells may include one or more enzymes capable of restrukturirovany tissue, in particular metalloprotease, semipretioase, neutral protease, hyaluronidase, RNase or Tnkase, etc. of Such enzymes include, but not limited to, collagenase (in particular, collagenase I, II, III or IV, or collagenase fromClostridium histolyticumand so on); dispute, thermolysin, elastase, trypsin, LIBERASE, hyaluronidase, etc.,

Composition for collection of cells may include bactericidal or bakteriostaticheski effective amount of an antibiotic. In some non-limiting embodiments, the present invention its implementation antibiotic is a macrolide (in particular, tobramycin), cephalosporins (in particular, cephalexin, cefuroxime, refrasil, cefaclor, cefixime, or cephalo-Smoking), clarithromycin, erythromycin, penicillin (in particular, penicillin V) or hinolan (in particular, ofloxacin, ciprofloxacin or norfloxacin, tetracycline, streptomycin, etc., In a specific embodiment, the present invention antibiotic active against gram(+) and/or gram(-) bacteria, includingPseudomonas aeruginosa,Staphylococcus aureusand so on

Composition for collecting cells may also include one or more of the following compounds: adenosine (from approximately 1 mm to approximately 50 mm); D-glucose (from priblisitelno mm to approximately 100 mm); magnesium ions (from approximately 1 mm to approximately 50 mm); in one embodiment of the present invention, the macromolecule with a molecular weight greater than 20,000 daltons present in sufficient quantity to maintain the integrity of the endothelium and the viability of the cells (in particular, synthetic or naturally occurring colloid, a polysaccharide such as dextran or polyethylene glycol, which is present in an amount of from about 25 g/l to about 100 g/l, or from about 40 g/l to about 60 g/l); antioxidant (in particular, butylsilane hydroxyanisol, butylsilane hydroxytoluene, glutathione, vitamin C or vitamin E, which is present in an amount of from about 25 microns to about 100 microns); a reducing agent (in particular, N-acetylcysteine, which is present in an amount of from about 0.1 mm to about 5 mm); an agent that prevents the release of calcium into the cells (in particular, verapamil, which is present in an amount of from about 2 microns to about 25 microns); nitroglycerin (for example, from about 0.05 g/l to about 0.2 g/l); in one embodiment of the present invention, the anticoagulant is present in a quantity sufficient to prevent clotting of residual blood (in particular, heparin or hirudin, which is resultsthe with a concentration of about 1000 units/l to about 100,000 units/l); or amyloidogenesis connection (in particular, amiloride, ethylisopropylamine, hexamethylenamine, dimethylaniline or isobutylamine, which is present in an amount of from approximately 1.0 μm to approximately 5 μm).

5.1.2. Collection of placental and manipulate it

In General, human placenta extract immediately after its removal after birth. In a preferred embodiment of the present invention the placenta is extracted from a patient after informed consent and after fully studied medical records of the patient associated with the placenta. Medical card continues to fill and after childbirth. Such medical card can be used to coordinate future use of the placenta or collected from her cell. For example, the cells of the human placenta can be used in accordance with the medical record, associated with the placenta personalized treatment baby, or parents, or brothers and sisters, or other relatives of the child.

Before removing the perfusion solution to remove blood from the umbilical cord blood and placenta. In some embodiments, implementation of the present invention after childbirth remove blood from the umbilical cord to the placenta. Cord blood from the placenta is removed in the usual way. To release the placenta from the blood slide is m, as a rule, use a needle or cannula (see, in particular, Anderson, U.S. patent No. 5372581; Hessel et al., U.S. patent No. 5415665). The needle or cannula is usually placed in the umbilical cord Vienna and the placenta may be gently massaged to facilitate drainage of umbilical cord blood from the placenta. Such extraction of umbilical cord blood can be done on a commercial basis, for example, LifeBank Inc., Cedar Knolls, N. J., ViaCord, Cord Blood Registry and CryoCell. Preferably, spend the drainage of the placenta by gravity, not through further manipulation in order to minimize the destruction of tissues in the process of extracting cord blood.

Typically, the placenta after delivery or transport from the delivery room to another location, in particular, to the laboratory for extraction of umbilical cord blood and collection of the perfusion solution. The placenta, preferably, transported in sterile, insulated transport device (the temperature of the placenta to maintain the temperature 20-28°C), for example, put the placenta with squeezed proximal end of the umbilical cord in a sterile package with zipper, which is then placed in an insulated container. In another embodiment of the present invention the placenta is transported using the kit for cord blood collection, as described in U.S. patent No. 7147626. The placenta is preferably transported to the laboratory within four is about twenty-four hours after birth. In some embodiments, implementation of the present invention before removing the cord blood proximal end of the umbilical cord pinch, preferably in the range of 4-5 cm from the exit site of the umbilical cord from the placenta rings. In other embodiments, implementation of the present invention the proximal end of the umbilical cord compress after extracting cord blood, but before further processing of the placenta.

Before collecting the placenta perfusion solution can be stored under sterile conditions either at room temperature or at a temperature of from 5 to 25°C. Before perfusion, in order to remove any residual cord blood, the placenta can be stored for more than forty-eight hours and preferably for from four to twenty-four hours. The placenta, mainly stored in the anticoagulant solution at a temperature in the range from 5 to 25°C. Suitable anticoagulant solutions are well known from the technical field. For example, you can use heparin or sodium derivative of warfarin. In a preferred embodiment of the present invention, the anticoagulant solution is a solution of heparin (in particular, 1% wt./mass. solution 1:1000). Before collecting placental perfusion solution released from the blood of the placenta, preferably, stored for a period of not more than 36 h..

5.1.3. PE is the fusion of the placenta

Methods perfusion of placental mammals are disclosed, in particular Hariri, U.S. patent No. 7045148 and 7255879, and in the patent application U.S. No. 2007/0190042 entitled "Improved composition for collecting and preserving bodies", the descriptions of which are entirely included in the present invention by reference.

The perfusion solution can be obtained by passing a solution for perfusion, in particular, saline, culture medium, or the above composition for collecting cells through the vasculature of the placenta. In one embodiment of the present invention the placenta of a mammal perfusion passing the solution for perfusion or through the umbilical artery or umbilical vein, or through the umbilical artery and the umbilical vein. The flow solution for perfusion through the placenta can be done, for example, by gravity through the placenta. The solution for perfusion, preferably forcibly pumped through the placenta using a pump, in particular a peristaltic pump. The umbilical vein can, in particular, to catheterizable using a cannula, such as cannula from Teflon or plastic that is attached to a sterile connecting device, such as a sterile tube. Sterile connecting device is connected to the reservoir for perfusion.

In the process of preparation for the perfusion of the placenta, preferably, ori is notroot thus, to umbilical artery or umbilical vein was located at the highest point of the placenta. The placenta can be perfusionist passing the solution for perfusion through the vasculature of the placenta or through the vasculature of the placenta and the surrounding tissue. In one embodiment, the present invention umbilical artery and the umbilical vein at the same time attached to the pipette, which through a flexible connecting device is connected to the vessel with a solution for perfusion. The solution for perfusion flow through the umbilical vein and artery. The solution for perfusion stands out from the blood vessels and/or passes through the walls of blood vessels into the surrounding tissue of the placenta and is collected in a suitable open vessel with the surface of the placenta, which is attached to the mother's uterus during pregnancy. The solution for perfusion can also enter through the hole in the umbilical cord and give it to flow and to seep out of the holes in the wall of the placenta, which was docked with the wall of the uterus of the mother. In another embodiment of the present invention the solution for perfusion passes through the umbilical vein and is collected from the umbilical artery, or passes through the umbilical artery and is collected from the umbilical vein, i.e., passes only through the vasculature of the placenta (fetal tissue).

In one embodiment of the present invention, for example, the R, the umbilical artery and the umbilical vein at the same time attach, in particular, to the pipette, which through a flexible connecting device is connected to the vessel with a solution for perfusion. The solution for perfusion flow through the umbilical vein and artery. The solution for perfusion stands out from the blood vessels and/or passes through the walls of blood vessels into the surrounding tissue of the placenta and is collected in a suitable open vessel with the surface of the placenta, which is attached to the mother's uterus during pregnancy. The solution for perfusion can also enter through the hole in the umbilical cord and give it to flow and to seep out of the holes in the wall of the placenta, which was docked with the wall of the uterus of the mother. Cells of the placenta collected in this way, which can be called the way “pallet” as a rule, are a mixture of fetal cells and cells of the mother.

In another embodiment of the present invention the solution for perfusion passes through the umbilical vein and is collected from the umbilical artery, or passes through the umbilical artery and is collected from the umbilical vein. Cells of the placenta collected in this way, which can be called as a method “closed circuit”, as a rule, almost exclusively represent the cells of the fetus.

In one of the embodiments of the present invention, perfuse is on the way closed circuit can hold the following way. Postpartum placenta receive within 48 h after birth. The umbilical cord pinch and cut off above the clamp. The umbilical cord can be discarded or processed to extract, for example, stem cells of the umbilical cord, and/or to process the membrane of the umbilical cord for receipt of the biomaterial. Amniotic membrane can be saved during perfusion or be separated from the chorion, for example, otstaiva it with your fingers. If the amniotic membrane is separated from the chorion prior to perfusion, it can, for example, dropped or subjected to processing, in particular, with the purpose of obtaining stem cells by enzymatic cleavage or to obtain, for example, the biomaterial of the amniotic membrane, in particular, biomaterial, which is described in patent application U.S. No. 2004/0048796. After the placenta purified from visually observed blood clots and residual blood, for example, by using sterile gauze, the vessels of the umbilical cord is cut, for example, by partial cutting of the membrane of the umbilical cord to open the cut umbilical cord. Vessels identify and reveal, for example, promoting private clamp type “crocodile” through the cut end of each vessel. Then in each of the arteries of the placenta insert a device, such as a plastic tube attached to the device for carrying out perfusion or PE is istoricheskom pump. The pump can be any pump suitable for this purpose are, for example, by a peristaltic pump. Then in the vein of the placenta insert a plastic tube connected to a sterile collection, in particular to a package for storing blood, such as a package for collecting blood with a capacity of 250 ml In the alternative, the tube connected to the pump, is inserted into a vein in the placenta, and the tube connected to the collector(s) are inserted into one or both of the arteries of the placenta. Then the placenta perfusion a certain volume of solution for perfusion, for example, using approximately 750 ml solution for perfusion. After that collect cell perfusion solution, for example, by centrifugation.

In one embodiment of the present invention the proximal end of the umbilical cord compress during perfusion and, more preferably, compress within 4-5 cm from the junction of the umbilical cord from the placenta ring.

The first collection of fluid perfusion of the placental mammal in the process of liberation from the blood is usually painted residual red blood cells cord blood and/or blood in the placenta. The fluid perfusion becomes more colorless as the perfusion and as residual umbilical cord blood cells are washed away from the placenta. Typically, from 30 to 100 ml solution for perfusion enough for the first you is ivania blood from the placenta, however, depending on the observed results may be more or less fluid perfusion.

The volume of fluid perfusion, which is used for perfusion of the placenta, can vary depending on the number of cells from the placenta, which need to be collected, the size of the placenta, the amount of fees that need to be performed for a single placenta, etc., In various embodiments, implementation of the present invention, the volume of fluid perfusion can be from 50 ml to 5000 ml, 50 ml to 4000 ml, 50 ml to 3000 ml from 100 ml to 2000 ml, 250 ml to 2000 ml, from 500 ml to 2000 ml or 750 ml to 2000 ml As a rule, after the liberation from the blood of the placenta perfusion using 700-800 ml of fluid for perfusion.

The placenta can be perfusionist many times within a few hours or a few days. In the case when the placenta is supposed to perfusionist many times, it can be maintained or grown in a nutrient medium under aseptic conditions in a suitable vessel and perfusionist composition for collecting cells or standard solution for perfusion (in particular, normal saline solution such as phosphate buffered saline (“PBS”), which contains or does not contain an anticoagulant (e.g. heparin sodium derived warfarin, coumarin, bishydroxycoumarin and/or which contains or does not contain an antimicrobial agent (e.g., β-mercaptoethanol (0.1 mm); antibiotics such as streptomycin (in particular, with a concentration of 40-100 μg/ml), penicillin (e.g., in the amount of 40 units/ml), amphotericin b (in particular, with a concentration of 0.5 μg/ml). In one embodiment of the present invention an isolated placenta maintain or grow in a nutrient medium for some time, not collecting perfusion solution, so that the placenta is maintained or grown in a nutrient medium within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24 h, or within 2 or 3 days prior to the perfusion and collection of the perfusion solution. The placenta after perfusion can be maintained during more or less long period of time, for example, within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or more hours and perfusionist a second time, using, for example, 700 to 800 ml of fluid for perfusion. The placenta can be perfusionist 1, 2, 3, 4, 5 or more times, for example, once every 1, 2, 3, 4, 5 or 6 o'clock In the preferred embodiment of the present invention perfusion of the placenta and collection solution for perfusion, for example, compositions for collecting placental cells, to repeat until the number of extracted nucleated cells falls below 100 cells/ml. Perfusate obtained at different points in time can be further processed individually is, to extract the time-dependent populations of cells, in particular, the aggregate nucleated cells. You can also combine into a common Fund perfusate obtained at different points in time.

5.1.4. Placental perfusion solution and cells placental perfusion solution

Placental perfusion solution is a heterogeneous set of cells. Typically, before using placental perfusion solution released from erythrocytes. Such exemption can be made using known methods of separating red blood cells from nucleated blood cells. In one embodiment of the present invention, the perfusion solution or cell perfusion solution stored at cryogenic temperatures. In some other embodiments, implementation of the present invention placental perfusion solution contains, or cells of placental perfusion solution represent only the cells of the fetus or a combination of fetal cells and cells of the mother.

Typically, the placental perfusion solution obtained after carrying out one of perfusion, contains from about 100 million to about 500 million nucleated cells. In some embodiments, implementation of the present invention placental perfusion solution or cells placental perfusion solution include cells CD34+in particular hematopoietic stem cells or cells of the predecessor is. Such cells can, in a more specific embodiment of the present invention, include CD34+CD45‾ stem cells or precursor cells, CD34+CD45+stem cells or precursor cells, myeloid precursors, lymphoid precursors and/or erythroid precursors. In other embodiments, implementation of the present invention placental perfusion solution or cells placental perfusion solution include adherent placenta stem-cells, in particular stem cells CD34‾. In another embodiment, the present invention placental perfusion solution or cells placental perfusion solution include, for example, endothelial precursor cells, osteoprogenitor cells and natural killer cells. In some embodiments, implementation of the present invention placental perfusion solution, after he separated from the placenta and released from red blood cells, or cells of the perfusion solution, isolated from similar perfusion solution includes approximately 6-7% of natural killer cells (CD3‾, CD56+); approximately 21-22% of T cells (CD3+); approximately 6-7% of b-cells (CD19+); approximately 1-2% of endothelial progenitor cells (CD34+, CD31+); approximately 2-3% neutral progenitor cells (nestin+); approximately 2-5% of hematopoietic cells preds the owners (CD34 +); and about 0.5-1.5% of adherent stem cells of the placenta (in particular, CD34‾, CD117‾, CD105+and CD44+) that determine, for example, by flow cytometry, in particular, the analysis by FACS method.

5.2. The destruction and breakdown of placental tissue to obtain PINK cells

Natural killer cells of the placenta, in particular PINK cells can be obtained from placental tissue, which is subjected to mechanical and/or enzymatic degradation.

Placental tissue can be restrukturirovany using one or more enzymes capable of restrukturirovany tissue, in particular metalloprotease, semipretioase, neutral protease, RNase or Tnkase, etc. of Such enzymes include, but not limited to, collagenase (in particular, collagenase I, II, III or IV, collagenase fromClostridium histolyticumand so on); dispute, thermolysin, elastase, trypsin, LIBERASE, hyaluronidase, etc., usually after fermentation subjected to fermentation fabric is passed through percolator or filter to remove partially fermented clumps of cells, while having almost single cell suspension.

After receiving the cell suspension placental natural killer cells can be isolated using, for example, antibodies to CD3 and CD56. In a specific embodiment, the present invention natural glue the key killers of the placenta is separated, selecting cells that are CD56+and get the first cell population; carry out the contacting of the specified first population of cells with antibodies specific for CD3 and/or CD16; and remove cells from the first specified population which constitute the CD3+or CD56+will get a second population of cells, which largely represent cells CD56+and CD3‾, CD56+and CD16‾ or CD56+, CD3‾ and CD16‾.

In one embodiment of the present invention for allocation of natural killer cells of the placenta from the suspension of placental cells using magnetic beads. Cells can be selected, for example, using methods magnitoekranirovannyih cell sorting (MACS), the method of separating particles based on their ability to bind magnetic beads (in particular, having a diameter of 0.5-100 μm), which include one or more specific antibodies, in particular antibodies against CD56. Magnetic microspheres are various acceptable methods of modify, including covalent addition of antibodies that recognize specific specific molecule or hapten on the surface of the molecule. The beads are then mixed with the cells to allow them to interact. The cells are then passed through a magnetic field, with the aim of identifying cells kotoryi.kot specific cell surface marker. In one embodiment of the present invention, these cells can be isolated and re-mixed with the magnetic beads bound with antibodies against additional markers on the cell surface. These cells are again passed through a magnetic field and isolate cells that bind both antibodies. Such cells can then be diluted into individual tablets, such as tablets for micrometrology to select the clones.

5.3. Natural killer cells of the placenta

In accordance with one aspect, the present invention proposes an allocation, the study of the characteristics and application of natural killer cells from the placenta, in particular, from placental perfusion solution and/or subjected to mechanical and/or enzymatic degradation of the tissue of the placenta, as well as compositions comprising such natural killer cells. In a specific embodiment, the present invention natural killer cells of the placenta are “assisted natural killer cells of the placenta” or “PINK” cells, which is characterized as cells CD56+CD16‾, meaning that cell marker CD56 and not having cell marker CD16, which define flow cytometry, for example, by the method of fluorescent-activated cell sorting using antibodies against CD16 and CD56, the AK described above. Thus, the present invention offers a dedicated PINK cells and the selected set of PINK cells. In addition, the present invention offers a selected variety of cells, including PINK cells CD56+CD16‾ combined with natural killer cells CD56+CD16+. In a more specific embodiment of the present invention natural killer cells CD56+CD16+can be isolated from the placenta or from another source such as peripheral blood, umbilical cord blood, bone marrow, etc. So, in various other embodiments, implementation of the present invention PINK cells can be combined with natural killer cells CD56+CD16+in particular, in ratios, for example, approximately equal to 1:10, 2:9, 3:8, 4:7, 5:6, 6:5, 7:4, 8:3, 9:2, 1:10, 1:9, 1:8, 1:7, 1:6, 1:5, 1:4, 1:3, 1:2, 1:1, 2:1, 3:1, 4:1, 5:1, 6: 1, 7:1, 8:1 or about 9:1. In the present description, the term “isolated” means that cells are removed from their environment, in particular from the placenta.

In some embodiments, implementation of the present invention PINK cells are CD3‾.

In other embodiments, implementation of the present invention PINK cells do not show one or more cellular markers that are fully Mature natural killer cells (in particular, CD16), or show such one or more Mar the development with a significantly reduced level, compared to fully Mature natural killer cells, or show one or more cellular markers associated with the precursor natural killer cells, but not fully Mature natural killer cells. In a specific embodiment, the present invention proposed in this invention PINK cells Express NKG2D, CD94 and/or NKp46 at a noticeably lower level than fully Mature PINK cells. In a more specific embodiment, the present invention proposed in the present invention many PINK cells expresses, in General, NKG2D, CD94 and/or NKp46 at a noticeably lower level than an equivalent number of fully Mature NK cells.

In some embodiments, implementation of the present invention PINK cells Express one or more molecules of the microRNA hsa-miR-100, hsa-miR-127, hsa-miR-211, hsa-miR-302c, hsa-miR-326, hsa-miR-337, hsa-miR-497, hsa-miR-512-3p, hsa-miR-515-5p, hsa-miR-517b, hsa-miR-517c, hsa-miR-518a, hsa-miR-518e, hsa-miR-519d, hsa-miR-520g, hsa-miR-520h, hsa-miR-564, hsa-miR-566, hsa-miR-618, and/or hsa-miR-99a with a significantly higher level than the natural killer cells in peripheral blood.

In some embodiments, implementation of the present invention natural killer cells of the placenta, in particular PINK cells, propagated in a nutrient medium. In some other embodiments, implementation of the present invention in a nutrient medium times nozaut cells placental perfusion solution. In a specific embodiment of the present invention, these cells are placental perfusion solution multiply in the presence of feeder and/or in the presence of at least one cytokine. In a more specific embodiment of the present invention the specified feeder includes cells C or mononuclear cells of peripheral blood. In another more specific embodiment, the present invention is specified, at least one cytokine is an interleukin-2.

In another embodiment, the present invention offers a selected variety (e.g., population) PINK cells. In another specific embodiment of the present invention the selected cell population obtained by isolating cells from placental perfusion solution using CD56-beads. In various specific embodiments, implementation of the present invention this population includes at least approximately 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or at least about 99% PINK cells. In another embodiment, the present invention plenty of PINK cells comprises, or consists of, PINK cells, which were multiplied; in particular, it is a lot of PINK cells in the form in which they are isolated from the placental perfusion solution. In another embodiment, the present image is the shadow of the many PINK cells includes, or consists of, PINK cells that were propagated. The methods of dissemination PINK cells are described, in particular, Ohno et al., in the application for U.S. patent No. 2003/0157713; see also Yssel et al.,J. Immunol. Methods72(1):219-227 (1984) and Litwin et al.,J. Exp. Med.178(4):1321-1326 (1993), and the method of reproduction of natural killer cells below in example 1.

In other embodiments, implementation of the present invention, the selected set PINK cells does not show one or more cellular markers that show a fully Mature natural killer cells (in particular, CD16), or shows one similar or more similar cellular markers on a significantly lower level compared to fully Mature natural killer cells, or shows one or more cellular markers associated with the precursor natural killer cells, but not associated with a fully Mature natural killer cells. In a specific embodiment, the present invention proposed in this invention PINK cells Express NKG2D, CD94 and/or NKp46 at a noticeably lower level than fully Mature NK cells. In a more specific embodiment, the present invention proposed in the present invention many PINK cells expresses, in General, NKG2D, CD94 and/or NKp46 at a noticeably lower level than an equivalent num is fully Mature NK cells.

In specific embodiments, the implementation of the present invention population of PINK cell expresses one or more molecules of the microRNA hsa-miR-100, hsa-miR-127, hsa-miR-211, hsa-miR-302c, hsa-miR-326, hsa-miR-337, hsa-miR-497, hsa-miR-512-3p, hsa-miR-515-5p, hsa-miR-517b, hsa-miR-517c, hsa-miR-518a, hsa-miR-518e, hsa-miR-519d, hsa-miR-520g, hsa-miR-520h, hsa-miR-564, hsa-miR-566, hsa-miR-618, and/or hsa-miR-99a with a significantly higher level than the natural killer cells in peripheral blood. In another specific embodiment, the present invention population of PINK cells expresses significantly higher number of granzyme In than the equivalent number of natural killer cells in peripheral blood.

In other embodiments, implementation of the present invention proposed in this invention PINK cells propagated in growth medium. In specific embodiments, the implementation of the present invention PINK cells are cultivated in a nutrient medium, in particular, propagated in a nutrient medium during at least or not more than, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27 or 28 days. In a specific embodiment, the present invention PINK cells propagated in growth medium for approximately 21 days.

In accordance with another embodiment of the present invention, it is proposed a selected population of cells, in particular cells of the placenta, including PINK cells. In the specific embodiment of the present invention the selected population of cells is a combination of nucleated cells from placental perfusion solution, in particular, the total number of cells placental perfusion solution comprising autologous highlighted in PINK cells. In another specific embodiment, the present invention population of cells represents a selected population of cells obtained by separation of the cells from placental perfusion solution using CD56-beads. In various specific embodiments, implementation of the present invention this population includes at least approximately 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or at least about 99% PINK cells.

Because postpartum placenta includes tissues and cells of the fetus and the mother cells, depending on the method of collecting placental perfusion solution may include only cells of the fetus or the vast majority of fetal cells (in particular, greater than about 90%, 95%, 98% or 99%) or may include a mixture of cells of the fetus and the mother (in particular, the cells of the fetus are less than approximately 90%, 80%, 70%, 60% or 50% of the total number of nucleated cells in the perfusion solution). In one embodiment, the present invention PINK cells emit only from cells of the placenta of the fetus, in particular cells are obtained by perfusion of the placenta by the method of closed loop (see above), while perfusion gives the perfusion solution, including the vast majority of cells in the placenta of the fetus or consisting only of cleto the placenta of the fetus. In another embodiment, the present invention PINK cells isolated from the cells of the fetus and the mother, in particular cells are obtained by perfusion according to the method of the pallet (see above), while perfusion gives the perfusion solution comprising a mixture of cells of the placenta of the fetus and the mother. Thus, in accordance with one embodiment of the present invention serves a population isolated from the placenta helper and natural killer cells, the vast majority of which has the phenotype of the fetus. In accordance with another embodiment of the present invention serves a population isolated from the placenta helper and natural killer cells, which includes natural killer cells having the phenotype of the fetus, and natural killer cells having the phenotype of the mother.

In the present invention are also populations isolated from placenta helper and natural killer cells, which include natural killer cells from non-placental source. For example, in one embodiment, the present invention serves a population of PINK cells, which also includes natural killer cells from umbilical cord blood, peripheral blood, bone marrow, or a combination of two or more of these sources. The population of natural killer cells, on the expectation PINK cells and natural killer cells from non-placental source, may contain cells, for example, in a ratio of about 1:10, 2:9, 3:8, 4:7, 5:6, 6:5, 7:4, 8:3, 9:2, 10:1, 1:9, 1:8, 1:7, 1:6, 1:5, 1:4, 1:3, 1:2, 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 100:1, 95:5, 90:10, 85:15, 80:20, 75:25, 70:30, 65:35, 60:40, 55:45, 50:50, 45:55, 40:60, 35:65, 30:70, 25:75, 20:80, 15:85, 10:90, 5:95, 100:1, 95:1, 90:1, 85:1, 80:1, 75:1, 70:1, 65:1, 60:1, 55:1, 50:1, 45:1, 40:1, 35:1, 30:1, 25:1, 20:1, 15:1, 10:1, 5:1, 1:1, 1:5, 1:10, 1: 15, 1:20, 1:25, 1:30, 1:35, 1:40, 1:45, 1:50, 1:55, 1:60, 1:65, 1:70, 1:75, 1:80, 1:85, 1:90, 1:95 or about 1:100, or etc.

In addition, the present invention offers a combination of umbilical cord blood and highlighted in PINK cells. In various embodiments, implementation of the present invention umbilical cord blood is combined with the PINK cells in the amount of approximately 1×104, 5×104, 1×105, 5×105, 1×106, 5×106, 1×107, 5×107, 1×108or 5×108or more PINK cells per ml of cord blood.

In the present invention are also emphases the PINK cells. In one embodiment, the present invention PINK cells are harvested by obtaining placental perfusion solution, contacting the placental perfusion solution with a composition that is specific binds cells CD56+in particular, with the antibody against CD56, followed by separation of the cells CD56+based on the specified binding, you get a population of cells CD56+. The population of cells CD56+represents vydeleny the th population of natural killer cells. In a specific embodiment, the present invention cells CD56+contact with the composition, which is associated with specific cells CD16+in particular, with the antibody against CD16, and cells CD16+from a population of cells CD56+. In another specific embodiment, the present invention cells CD3+also excluded from the population of cells CD56+.

In one embodiment, the present invention PINK cells are obtained from placental perfusion solution as follows. The human placenta after giving birth free from the blood and perfusion, in particular, using approximately 200-800 ml solution for perfusion, only through the vasculature of the placenta. In a specific embodiment of the present invention provide umbilical cord blood to drain and washed vasculature of the placenta, in particular, with a solution for perfusion, to remove any residual erythrocytes. The perfusion solution is collected and processed, in order to remove any residual erythrocytes. Natural killer cells from the total number of nucleated cells in the perfusion solution can be distinguished based on the expression of CD56 and CD16. In some embodiments, implementation of the present invention the selection PINK cells enables selection using antibodies against CD56, with the selected cells are CD56+. the other embodiment of the present invention the selection PINK cells enables selection using antibodies against CD16, when the selected cells are CD16‾. In another embodiment, the present invention highlighting PINK cells enables selection using antibodies against CD56, and the elimination of many cells other than PINK cells, using antibodies against CD16, with the selected cells include cells CD56+, CD16‾.

Cells can be distinguished by any method known from the technical field, in particular by the method of fluorescent-activated cell sorting (FACS) or, preferably, by the method of magnetic cell sorting using beads that are associated with specific antibodies. Magnetic separation of cells can be done in automatic mode, using, for example, the separator AUTOMACS™ Separator (Miltenyi).

In accordance with another aspect, the present invention proposes a method of allocation of natural killer cells of the placenta, which includes many of the cells of the placenta and the Department of natural killer cells from the specified set of cells of the placenta. In a specific embodiment, the present invention placental cells are or cells of the placenta are cells of the placental perfusion solution, in particular, the combination of nucleated cells from placental perfusion solution. In another specific embodiment, the present invention KL is DAMI placenta are or cells of the placenta are cells of the placenta, the mechanical and/or enzymatic degradation of placental tissue. In another embodiment of the present invention the specified highlighting is performed using one or more antibodies. In a more specific embodiment of the present invention mentioned one antibody or several antibodies represent one or more antibodies against CD3, CD16 or CD56. In a more specific embodiment of the present invention the specified selection includes separation of cells CD56+from cells CD56‾ in a specified set of cells of the placenta. In a more specific embodiment of the present invention the specified selection includes separation of the placental cells CD56+, CD16‾, such as natural killer cells of the placenta, in particular PINK cells from placental cells that are CD56‾ or CD16+. In a more specific embodiment of the present invention the specified selection includes separation of the placental cells CD56+, CD16‾, CD3‾ from placental cells that are CD56‾, CD16+or CD3+. In another embodiment, the present invention this method of allocation of natural killer cells of the placenta leads to a population of placental cells that, at least on 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or, at least 99% consists of nature is lnyh killer cells CD56 +, CD16‾.

5.4. Natural killer cells of the placenta from compatible with each other perfusion solution and umbilical cord blood

In addition, the present invention offers natural killer cells, which emit or may be selected from a combination of matching samples of placental perfusion solution and umbilical cord blood, which in the present description is designated as a combined natural killer cells. The term “compatible samples” in this description means that natural killer cells are derived from cells of placental perfusion solution, cells in umbilical cord blood, umbilical cord blood cells obtained from umbilical cord blood, placenta, from which the placental perfusion solution, i.e., from the cells of placental perfusion solution and cells in umbilical cord blood, and, therefore, natural killer cells from each source belong to the same individual.

In some embodiments, implementation of the present invention combined natural killer cells of the placenta are just natural killer cells or virtually represent only natural killer cells, which are cells CD56+and CD16‾. In some other embodiments, implementation of the present invention combined natural killer cells of the placenta are natural CL the TCI-killers, which are the cells CD56+and CD16‾, and natural killer cells, which are cells CD56+and CD16+. In some specific embodiments, the implementation of the present invention combined natural killer cells of the placenta, at least 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 99,5% are natural killer cells CD56+CD16‾ (PINK cells).

In one embodiment of the present invention combined natural killer cells of the placenta are not grown in a nutrient medium. In a specific embodiment, the present invention combined natural killer cells include detective more natural killer cells CD3‾CD56+CD16‾ than an equivalent number of natural killer cells from peripheral blood. In another specific embodiment, the present invention combined natural killer cells include detective fewer natural killer cells CD3‾CD56+CD16‾ than an equivalent number of natural killer cells from peripheral blood. In another specific embodiment, the present invention combined natural killer cells include detective more natural killer cells CD56+KIR2DL2/L3+than an equivalent number of natural the s killer cells from peripheral blood. In another specific embodiment, the present invention combined natural killer cells include detective fewer natural killer cells CD3‾CD56+NKp46+than an equivalent number of natural killer cells from peripheral blood. In another specific embodiment, the present invention combined natural killer cells include detective fewer natural killer cells CD3‾CD56+NKp30+than an equivalent number of natural killer cells from peripheral blood. In another specific embodiment, the present invention combined natural killer cells include detective fewer natural killer cells CD3‾CD56+2B4+than an equivalent number of natural killer cells from peripheral blood. In another specific embodiment, the present invention combined natural killer cells include detective fewer natural killer cells CD3‾CD56+CD94+than an equivalent number of natural killer cells from peripheral blood.

In another embodiment, the present invention combined natural killer cells of the placenta are cultivated in a nutrient medium, for example, within 21 days. In the context of the specific embodiment of the present invention combined natural killer cells include detective fewer natural killer cells CD3‾CD56 +KIR2DL2/L3+than an equivalent number of natural killer cells from peripheral blood. In another specific embodiment, the present invention combined natural killer cells of the placenta are not grown in a nutrient medium. In another specific embodiment, the present invention combined natural killer cells include detective more natural killer cells CD3‾CD56+NKp44+than an equivalent number of natural killer cells from peripheral blood. In a specific embodiment, the present invention combined natural killer cells include detective more natural killer cells CD3‾CD56+NKp30+than an equivalent number of natural killer cells from peripheral blood.

In another embodiment, the present invention combined natural killer cells Express detective more granzyme In than the equivalent number of natural killer cells from peripheral blood.

In addition, the present invention offers a combination of umbilical cord blood and combined natural killer cells. In various embodiments, implementation of the present invention umbilical cord blood combine with the combined genuine cowhide leather-makes the mi killer cells in the amount of approximately 1×10 4, 5×104, 1×105, 5×105, 1×106, 5×106, 1×107, 5×107, 1×108, 5×108combined natural killer cells per ml of cord blood.

5.5. The combination of perfusion solution/cells

In addition to placental perfusion solution, cells placental perfusion solution, combined natural killer cells, in particular supporting natural killer cells of the placenta, the present invention provides a composition comprising a perfusion solution or cells, for the purpose of suppressing the proliferation of tumor cells or a variety of tumor cells.

5.5.1. Combination placental perfusion solution, cell perfusion solution and selected from the placenta helper and natural killer cells

In the present invention are also compositions comprising combinations of placental perfusion solution, cells placental perfusion solution, supporting natural killer cells of the placenta and/or combined natural killer cells above in sections 5.1, 5.3 or 5.4. In one of the embodiments of the present invention is proposed, for example, a certain amount of placental perfusion solution, supplemented by a variety of cells placental perfusion solution and/or a variety of natural killer cells of the placenta, in particular supporting natural cell-Ki is lerow placenta, for example, derived from cells of placental perfusion solution or placental tissue, subjected to mechanical and/or enzymatic degradation. In specific embodiments, the implementation of the present invention, for example, each ml of placental perfusion solution is supplemented by adding approximately 1×104, 5×104, 1×105, 5×105, 1×106, 5×106, 1×107, 5×107, 1×108, 5×108or more cells of placental perfusion solution, supporting natural killer cells of the placenta and/or combined natural killer cells. In another embodiment of the present invention the set of cells of placental perfusion solution complement placental perfusion solution, supporting natural killer cells of the placenta and/or combined natural killer cells. In another embodiment, the present invention many of the supporting natural killer cells of the placenta Supplement placental perfusion solution, the cells of placental perfusion solution and/or combined natural killer cells. In specific embodiments, the implementation of the present invention, when additions using the perfusion solution, the volume of perfusion solution is approximately approximately greater than or approximately less than 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 8%, 6%, 4%, 2% or 1% of observationsto cells (in solution) plus the perfusion solution. In some other embodiments, implementation of the present invention, when cells placental perfusion solution combine with lots of PINK cells and/or combined natural killer cells, cells of placental perfusion solution is usually approximately approximately greater than or approximately less than 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 8%, 6%, 4%, 2% or 1% of the total number of cells. In some other embodiments, implementation of the present invention, when the PINK cells, combined with the many cells of placental perfusion solution and/or combined natural killer cells, PINK cells typically make up approximately approximately greater than or approximately less than 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 8%, 6%, 4%, 2% or 1% of the total number of cells. In some other embodiments, implementation of the present invention, when combined natural killer cells combine with PINK cells and/or cells of placental perfusion solution, combined natural killer cells typically make up approximately approximately greater than or approximately less than 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 8%, 6%, 4%, 2% or 1% of the total number of cells. In some other embodiments, implementation of the present invention, when the PINK cells, combined natural killer cells or cells of placental perfusion solution used to Supplement the texts placental perfusion solution, the volume of solution (in particular, saline, culture medium, etc.,) in which is suspended cells, approximately, approximately greater than or approximately less than 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 8%, 6%, 4%, 2% or 1% of the total volume of perfusion solution plus cells, where PINK cells before addition suspended approximately in the amount of 1×104, 5×104, 1×105, 5×105, 1×106, 5×106, 1×107, 5×107, 1×108, 5×108or more cells per milliliter.

In other embodiments, implementation of the present invention any of the above combinations, in turn, combine with umbilical cord blood, or nucleated cells from umbilical cord blood.

In addition, in the present invention serves the United placental perfusion solution, which is obtained from two or more sources, in particular from two or more placentas, and unite, i.e. receive the General Fund. Such a combined perfusion solution may include an approximately equal volume of perfusion solution from each source, or may include different amounts from each source. The relative amounts from each source, you can choose randomly or selected based on, for example, the concentration or amount of one or more cellular factors, particularly cytokines, facto the economical growth, hormones and so on; the number of placental cells in the perfusion solution from each source; or other characteristics of the perfusion solution from each source. Similarly to the General Fund may be merged perfusate multiple perfusion of the same placenta.

Similarly, the present invention provides cells placental perfusion solution and selected from the placenta helper and natural killer cells, which are derived from two or more sources, in particular from two or more placentas, and unite in the General Fund. This combined cell may include an approximately equal number of cells from two or more sources, or a different number of cells from two or more United in the General Fund sources. The relative number of cells from each source can be selected based on, for example, one or more specific cell types in the cells from which it is supposed to form a common Fund, for example, the number of cells CD34+the number of cells CD56+and so on

Pooled funds may include, in particular, placental perfusion solution, supplemented cells placental perfusion solution; placental perfusion solution, supplemented selected from the placenta helper and natural killer cells (PINK); placental perfusion solution, supplemented as CL is DAMI placental perfusion solution, and PINK cells; cells of placental perfusion solution, supplemented with placental perfusion solution; cells placental perfusion solution, supplemented PINK cells; cells of placental perfusion solution, supplemented as placental perfusion solution, and PINK cells; PINK cells supplemented with placental perfusion solution; PINK cells, supplemented cells placental perfusion solution; or PINK cells, as supplemented by the cells of the placental perfusion solution, and placental perfusion solution.

In addition, in the present invention serves placental perfusion solution, the cells of placental perfusion solution and support natural killer cells of the placenta, and their pooled funds or their combinations, which are analyzed to determine the degree or amount of inhibition of tumor (i.e., effectiveness), which can be expected, in particular, by adjusting the amount of placental perfusion solution or PINK cells, or a given volume of perfusion solution. For example, an aliquot or sample with a certain number of cells injected in contact with a known number of tumor cells in conditions that otherwise tumor cells could proliferate, and the rate of proliferation of tumor cells in the presence of placental perfusion solution, cell perfusion solution, natural killer cells of the placenta or combinations thereof in certain periods of time (in particular, the past is under 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 weeks or longer) compared to the proliferation equivalent to the number of tumor cells in the absence of perfusion solution, cell perfusion solution, natural killer cells of the placenta or combinations thereof. The effectiveness of placental perfusion solution, cell perfusion solution and/or PINK cells, or combinations thereof, or their combined funds can be expressed, for example, as the number of cells or amount of solution required to inhibit the growth of tumor cells, in particular approximately 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50% and so on

In some embodiments, implementation of the present invention placental perfusion solution, the cells of placental perfusion solution and PINK cells are proposed as units of pharmaceutical quality, which can be assigned to people. Such units may be offered in the form of a specific amount, for example, 100 ml, 150 ml, 200 ml, 250 ml, 300 ml, 350 ml, 400 ml, 450 ml, 500 ml or so on, this unit may be offered in such a way that they contained a specific number, for example, cells of placental perfusion solution, supporting natural killer cells of the placenta or both, in particular, 1×104, 5×104, 1×105, 5×105, 1×106, 5×106, 1×107, 5×107, 1×108, 5×108or more cells per milliliter, or 1×104, 5×104, 1×105, 5×105, 1×106, 5×106, 1×1 7, 5×107, 1×108, 5×108, 1×109, 5×109, 1×1010, 5×1010, 1×1011or more cells per unit. Such units may be offered in such a way that they contain a specific quantity of one of the two, or one of the three components, including placental perfusion solution, the cells of placental perfusion solution and/or PINK cells.

In the above combination placental perfusion solution, cells placental perfusion solution and/or PINK cells of any of the above, any two of these or all three of these placental perfusion solution, cells placental perfusion solution and/or PINK cells can be autologous with respect to the recipient (i.e., obtained from the recipient), or may be homologous with respect to the recipient (i.e., obtained from at least one other individual other than the recipient).

Any of the above or combinations of any of the above funds PINK cells, placental perfusion solution and/or placental perfusion solution may include natural killer cells CD56+CD16+for example, placental perfusion solution, peripheral blood, umbilical cord blood, bone marrow, etc., In specific embodiments, the implementation of the present invention combinations include approximately at least about, or no more than about 1×1045×10 4, 1×105, 5×105, 1×106, 5×106or more like natural killer cells per milliliter, or 1×104, 5×104, 1×105, 5×105, 1×106, 5×106, 1×107, 5×107, 1×108, 5×108, 1×109, 5×109, 1×1010, 5×1010, 1×1011or more cells per unit. Natural killer cells CD56+CD16+can be used in the form in which they isolated from a natural source, or can be grown before turning into one of the above combinations or pooled funds. Cells CD56+CD16+can be autologous with respect to the recipient (i.e., obtained from the same individual, and that placental perfusion solution, the cells of placental perfusion solution and/or PINK cells; or received from the individual), or may be homologous with respect to the recipient (i.e., obtained from the individual, different from the origin of placental perfusion solution, cells placental perfusion solution and/or PINK cells; or from the individual who is not a recipient).

Preferably, each unit is labeled with specific volume, cell number, cell type, and whether these units are enriched in specific cell types, and/or the effectiveness of a given number of cells in the unit, or showing how many milliliters unit causes the t suppression of proliferation of a particular type or types of tumor cells, which can be measured.

In the present invention are also compositions comprising an auxiliary natural killer cells, alone or in combination with cells of placental perfusion solution and/or placental perfusion solution. Thus, in accordance with another aspect, the present invention provides a composition that includes selected natural killer cells CD56+, CD16‾, these natural killer cells isolated from placental perfusion solution and the natural killer cells constitute at least 50% of the cells in the composition. In a specific embodiment of the present invention these natural killer cells constitute at least 80% of the cells in the composition. In another specific embodiment, the present invention specified composition includes selected natural killer cells CD56+, CD16+. In a more specific embodiment of the present invention these natural killer cells CD56+, CD16+obtained from another individual than these natural killer cells CD56+, CD16‾. In another specific embodiment of the present invention these natural killer cells obtained from the same individual. In a more specific embodiment, this is subramania these selected natural killer cells are natural killer cells, obtained from at least two different individuals. In another specific embodiment of the present invention, the composition includes a dedicated placental perfusion solution. In a more specific embodiment of the present invention specified placental perfusion solution obtained from the same individual, and that these natural killer cells. In a more specific embodiment of the present invention specified placental perfusion solution is a placental perfusion solution, obtained from a different individual than these natural killer cells. In another specific embodiment of the present invention the composition comprises cells of placental perfusion solution. In a more specific embodiment of the present invention, these cells are placental perfusion solution obtained from the same individual, and that these natural killer cells. In a more specific embodiment of the present invention, these cells are placental perfusion solution obtained from another individual than these natural killer cells. In another specific embodiment of the present invention, the composition additionally includes a dedicated placental perfusion solution and the selected cells placental perfusion solution, with the specified selected placental p is husat and these selected cells placental perfusion solution obtained from different individuals. In another more specific embodiment of embodiment of any of the above embodiments of the present invention, including placental perfusion solution specified placental perfusion solution is a placental perfusion solution, obtained from at least two individuals. In another more specific embodiment of embodiment of any of the above embodiments of the present invention, including cells of placental perfusion solution, these selected cells placental perfusion solution obtained from at least two individuals.

5.2.2. Compositions comprising adherent placenta stem-cells

In other embodiments, implementation of the present invention placental perfusion solution, the set of cells of placental perfusion solution and/or a lot of PINK cells, or combinations or pooled funds in any of these substances, Supplement adherent stem cells of the placenta. Such stem cells are described, for example, issued in the name of Hariri U.S. patents No. 7045148 and 7255879. Adherent stem cells from the placenta are not trophoblasts.

Placental perfusion solution, the set of cells of placental perfusion solution and/or a lot of PINK cells, or combinations or pooled funds in any of these substances can be added, for example, using 1×104, 5×104, 1×105, 5×105, 1×10 6, 5×106, 1×107, 5×107, 1×108, 5×108or more cells per milliliter, or 1×104, 5×104, 1×105, 5×105, 1×106, 5×106, 1×107, 5×107, 1×108, 5×108, 1×109, 5×109, 1×1010, 5×1010, 1×1011or more adherent cells of placenta. Adherent stem cells from the placenta in combination can be, for example, adherent stem cells of the placenta, which is grown in a nutrient medium, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38 or 40 population doublings or more.

Adherent placenta stem-cells, when cultured in primary cultures or in cell culture, adhere to the tissue culture substrate, in particular, to the surface of the container with tissue culture (for example, to plastic tissue culture). Adherent placenta stem-cells in culture usually take fibroblastoid, a star shape, wherein the main cells leaves a number of cytoplasmic appendages. However, adherent placenta stem-cells morphologically distinguishable from fibroblasts cultured under the same conditions, because the stem cells from the placenta receives a greater number of such appendages than fibroblasts. Morphologically stem cells from the placenta distinct from hematopoietic STV is gross cell which usually take smoother, or boleznetvornogo, morphology in culture.

Adherent stem cells from the placenta and the population of stem cells of the placenta, suitable for use in the compositions and methods of the present invention, Express many of the markers that can be used to identify and/or hematopoietic stem cells or populations of cells that comprise the stem cells. Adherent stem cells from the placenta and the population of adherent stem cells suitable for use in the compositions and methods of the present invention include stem cells and a population containing stem cells, which are obtained directly from the placenta or from any part of it (for example, from the amniotic membrane, chorion, amnion-chorionic plate, cotyledons placenta, umbilical cord, etc). In one embodiment, the present invention population of adherent stem cells of the placenta is the population (i.e., represents two or more adherent stem cells of the placenta in culture, for example, the population in the container, in particular in the package.

Adherent placenta stem-cells usually Express the markers CD73, CD105, CD200, HLA-G and/or OCT-4 and does not Express CD34, CD38, or CD45. Adherent stem cells from the placenta can also xpresroute HLA-ABC (MHC-1) and HLA-DR. These markers can be used to identify adherent stem cells of the placenta and to separate the stem cells of the placenta from other types of stem cells. Since stem cells from the placenta can Express CD73 and CD105, they can have properties similar to stem cells in the mesenchyme. However, since the adherent stem cells from the placenta can Express CD200 and HLA-G-specific fetal marker, they can be separated from the stem cells of the mesenchyme, in particular, bone-marrow-derived stem cells of the mesenchyme, which does not Express any CD200 or HLA-G. Similarly, the lack of expression of CD34, CD38 and/or CD45 identifies adherent stem cells from the placenta as not hematopoietic stem cells.

In one embodiment, the present invention adherent stem cells, placental cells are CD200+HLA-G+while stem cells markedly suppress cancer cell proliferation or tumor growth. In a specific embodiment, the present invention specified adherent stem cells are CD73+and CD105+. In another specific embodiment, the present invention specified adherent stem cells are also CD34‾, CD38‾ or CD45‾. In a more specific embodiment, implementation is tvline of the present invention are listed adherent stem cells are also CD34‾, CD38‾ and CD45‾, CD73+and CD105+. In another embodiment, the present invention indicated adherent stem cells produce one or more embryopathy Taurus, if they are grown in a nutrient medium under conditions that allow formation embryopathy calves.

In another embodiment, the present invention adherent stem cells, placental cells are CD73+, CD105+, CD200+while these stem cells markedly suppress cancer cell proliferation or tumor growth. In a specific embodiment of the above populations of these adherent stem cells are HLA-G+. In another specific embodiment, the present invention specified adherent stem cells are CD34‾, CD38‾ or CD45‾. In another specific embodiment, the present invention specified adherent stem cells are CD34‾, CD38‾ and CD45‾. In a more specific embodiment of the present invention are listed adherent stem cells are CD34‾, CD38‾, CD45‾ and HLA-G+. In another specific embodiment, the present invention indicated adherent placenta stem-cells produce one or more embryopathy Taurus, if they are grown in nutrient media is in terms which allow to form embryopathy calves.

In another embodiment, the present invention adherent stem placental cells are CD200+, OCT-4+while these stem cells markedly suppress cancer cell proliferation or tumor growth. In a specific embodiment, the present invention specified adherent stem cells are CD73+and CD105+. In another specific embodiment, the present invention specified adherent stem cells are HLA-G+. In another specific embodiment, the present invention specified adherent stem cells are CD34‾, CD38‾ and CD45‾. In a more specific embodiment of the present invention are listed adherent stem cells are CD34‾, CD38‾, CD45‾, CD73+, CD105+and HLA-G+. In another specific embodiment, the present invention adherent stem cells produce one or more embryopathy Taurus, if they are grown in a nutrient medium under conditions that allow formation embryopathy calves.

In another embodiment, the present invention adherent stem placental cells are CD73+, CD105+and HLA-G+when atomoxetinee adherent stem cells markedly suppress cancer cell proliferation or tumor growth. In a particular variant embodiment of the above many of these cells adherent stem cells are also CD34‾, CD38‾ or CD45‾. In another specific embodiment, the present invention specified adherent stem cells are also CD34‾, CD38‾ and CD45‾. In another specific embodiment, the present invention specified adherent stem cells are OCT-4+. In another specific embodiment, the present invention specified adherent stem cells are CD200+. In a more specific embodiment of the present invention are listed adherent stem cells are also CD34‾, CD38‾, CD45‾, OCT-4+and CD200+.

In another embodiment, the present invention adherent stem cells, placental stem cells are CD73+, CD105+while these stem cells produce one or more embryopathy Taurus under conditions that allow formation embryopathy calves, and these adherent stem cells markedly suppress cancer cell proliferation or tumor growth. In a specific embodiment, the present invention specified adherent stem cells are also CD34‾, CD38‾ and the and CD45‾. In another specific embodiment, the present invention specified adherent stem cells are also CD34‾, CD38‾ and CD45‾. In another specific embodiment, the present invention specified adherent stem cells are OCT-4+. In a more specific embodiment of the present invention are listed adherent stem cells are OCT-4+, CD34‾, CD38‾ and CD45‾.

In another embodiment, the present invention adherent stem cells, placental stem cells are OCT-4+while these stem cells from the placenta produce one or more embryopathy cells when they are grown in a nutrient medium under conditions that allow to form embryopathy calves, and these stem cells are identified as cells, markedly suppress cancer cell proliferation or tumor growth.

In various embodiments, implementation of the present invention, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or at least 95% of these selected stem cells of the placenta are stem cells OCT4+. In particular VA is iante embodiment of the above populations of these stem cells are CD73 +and CD105+. In another specific embodiment of the present invention these stem cells are CD34‾, CD38‾ or CD45‾. In another specific embodiment of the present invention these stem cells are CD200+. In a more specific embodiment of the present invention these stem cells are CD73+, CD105+, CD200+, CD34‾, CD38‾ and CD45‾. In another specific embodiment, the present invention specified population multiply, for example, subcultured cell culture medium at least once, at least three times, at least five times, at least 10 times, at least 15 times or at least 20 times.

In a more specific embodiment of any of the above embodiments of the present invention adherent stem cells from the placenta Express ABC-p (specific placental transport protein ABC; see, in particular, Allikmets et al.,Cancer Res.58(23):5337-9 (1998)).

In another embodiment, the present invention adherent stem cells of the placenta are CD29+, CD44+, CD73+, CD90+, CD105+, CD200+, CD34‾ and CD133‾. In another embodiment, the present invention adherent stem cells from the placenta, stem cells placenta Nera is wiremu secrete IL-6, IL-8 and macrophage chemoattractant protein (MCP-1).

Each of these stem cells placenta may include stem cells from the placenta, received and allocated directly from the placenta of a mammal, or stem cells from the placenta, which is grown in a nutrient medium and are subcultured on the medium, at least, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 25, 30 or more times, and their combinations. The above set of adherent stem cells of the placenta, inhibit tumor cells, may include about at least or not more than 1×105, 5×105, 1×106, 5×106, 1×107, 5×107, 1×108, 5×108, 1×109, 5×109, 1×1010, 5×1010, 1×1011or more adherent stem cells of the placenta.

5.5.3. Composition, including the environment, air-conditioned stem cells placenta

The present invention also proposes the use of oppressive tumor compositions that contain PINK cells, placental perfusion solution and/or placental perfusion solution and additionally air-conditioned environment. Adherent stem cells from the placenta, cells placental perfusion solution and/or auxiliary natural killer cells of the placenta can be used to obtain the air-conditioned environment, which are able to inhibit tumor cells, those. environment that includes one or more biomolecules secreted or produced by stem cells, which have detective suppressive effect on tumor cells, many of the immune cells of the same or of different types. In various embodiments, implementation of the present invention, air-conditioned environment is an environment in which cells of the placenta (in particular, stem cells, cells of placental perfusion solution, PINK cells) grown at least within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or more days. In other embodiments, implementation of the present invention, air-conditioned environment is an environment in which cells of the placenta grown, at least to the extent of merging 30%, 40%, 50%, 60%, 70%, 80%, 90% or until the degree of fusion of 100%. Such air-conditioned environment can be used to maintain the culture of a population of placental cells or cells of another species. In another embodiment, the present invention proposed in the present invention, air-conditioned environment is an environment in which grown adherent stem cells from the placenta and cells other than stem cells of the placenta.

Such air-conditioned environment can be combined with any combination of or any combination of placental perfusion solution, cells Platz is Tarnovo perfusion solution and/or auxiliary natural killer cells of the placenta with the formation of the composition, painful tumor cells. In specific embodiments, the implementation of the present invention, the composition includes less than half of the air-conditioned environment by volume, in particular, about or less than about 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2% or 1% by volume.

Thus, in one embodiment, the present invention features a composition comprising culture medium from the culture of stem cells of the placenta, where these stem cells (a) adhere to a substrate; (b) Express CD200 and HLA-G, or Express CD73, CD105, and CD200, or Express CD200 and OCT-4, or Express CD73, CD105, and HLA-G, or Express CD73 and CD105 and facilitate the formation of one or more embryopathy Taurus in a population of placental cells that comprise the stem cells from the placenta, when the specified population is grown in a nutrient medium under conditions that allow to form embryopathy Taurus, or Express OCT-4 and facilitate the formation of one or more embryopathy Taurus in a population of placental cells that comprise the stem cells from the placenta, when the specified population is grown in a nutrient medium under conditions that allow to form embryopathy Taurus; and (c) significantly inhibit the growth or proliferation of a tumor cell or population of tumor the x cells. In a specific embodiment of the present invention the composition also contains many of these stem cells placenta. In another specific embodiment of the present invention the composition contains many cells other than stem cells of the placenta. In a more specific embodiment of the present invention, these cells other than stem cells of the placenta include cells CD34+in particular, the precursor cells of hematopoietic cells, such as precursor cells of hematopoietic peripheral blood cells, or precursor cells of hematopoietic cells of placental blood. Cells other than stem cells of the placenta, also include other stem cells, such as stem cells of the mesenchyme, in particular, isolated from bone marrow stem cells of the mesenchyme. Cells other than stem cells of the placenta may also represent one or more types of Mature cells or cell lines. In another specific embodiment of the present invention, the composition comprises an antiproliferative agent, in particular an antibody against MIP-1α or against MIP-1β.

In a specific embodiment of the present invention the culture medium conditioned by placental cells or supernatant obtained from a variety of STW, the new cells of the placenta, grown in a nutrient medium in conjunction with a variety of tumor cells in a ratio of about 1:1, about 2:1, about 3:1, about 4:1 or about 5:1 stem cells placenta to tumor cells. For example, the conditioned culture medium or supernatant can be obtained from the culture containing approximately 1×105stem cells of the placenta, approximately 1×106stem cells of the placenta, approximately 1×107stem cells of the placenta, or about 1×108stem cells of the placenta or more. In a specific embodiment of the present invention, air-conditioned culture medium or supernatant from a culture containing approximately 1×105to approximately 5×105stem cells of the placenta and from about 1×105tumor cells; from about 1×106to approximately 5×106stem cells of the placenta and approximately 1×106tumor cells; from about 1×107to approximately 5×107stem cells of the placenta and approximately 1×107tumor cells; or from about 1×108to approximately 5×108stem cells of the placenta and approximately 1×108tumor cells.

In a specific embodiment, altoadige of the invention, air-conditioned environment, suitable for administration to an individual weighing 70 kg, is a supernatant, air-conditioned approximately 70 million stem cells of the placenta, approximately 200 ml of culture medium.

Air-conditioned environment can be concentrated, to get suitable for the introduction of pharmaceutical product. For example, an air-conditioned environment, you can concentrate about 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10% or more by removing water, in particular by evaporation, lyophilization, etc., In a particular embodiment of the present invention, for example, 200 ml of conditioned medium from approximately 70 million stem cells of the placenta can be concentrated up to a volume of approximately 180 ml, 160 ml, 140 ml, 120 ml, 100 ml, 80 ml, 60 ml, 40 ml, 20 ml or less. Air-conditioned environment, you can almost be dried, for example, to turn in powder, in particular by evaporation, lyophilization, etc.,

5.6. The preservation perfusion solution and cells of the placenta

Placental perfusion solution or cells of the placenta, in particular cells of the perfusion solution, combined natural killer cells, and PINK cells, can be saved, i.e., to put in terms that allow their long-term storage, or in conditions in which inhibited cell death, in particular, due to apoptosis or necrosis.

Placental perfuse which can be obtained by passing the composition for collecting placental cells, at least through part of the placenta, in particular, through the vasculature of the placenta. Composition for collecting placental cells includes one or more compounds that have a preservative effect on contained in the perfusion solution cells. A similar arrangement for collecting placental cells described above in section 5.1, in particular, it can be a composition that includes an inhibitor of apoptosis inhibitor, necrosis and/or perfluorocarbons, able to carry oxygen, as described in related application U.S. patent No. 11/648812, entitled “Improved composition for collecting and preserving stem cells of the placenta and the method of applying the specified composition, which filed December 28, 2006, In one embodiment of the present invention, the composition for collecting placental cells, passed through the placenta or placenta tissue, represents the perfusion solution, suitable for use in the methods described below in section 5.4.

In one embodiment, the present invention placental perfusion solution and/or cells of the placenta are collected from a postpartum placenta of a mammal, in particular human, by contacting the cells with a composition for collecting placental cells that comprises an inhibitor of apoptosis and perfluorocarbons, able to carry oxygen, with the indicated inhibitor of apoptosis when outstay in the quantities and within the time sufficient to reduce or prevent apoptosis in the population of stem cells, compared with the population of stem cells that are not contacted with the inhibitor of apoptosis. For example, the placenta can be perfusionist composition for collecting placental cells and to extract the cells of the placenta, for example, the combination of nucleated cells of the placenta. In a specific embodiment of the present invention the inhibitor of apoptosis is a caspase inhibitor. In another specific embodiment of the present invention the inhibitor of apoptosis is a JNK inhibitor. In a more specific embodiment of the present invention specified JNK inhibitor does not regulate the differentiation or proliferation of these stem cells. In another embodiment of the present invention, the composition for collecting placental cells contains the specified inhibitor of apoptosis and the specified perfluorocarbons, able to carry oxygen, in different phases. In another embodiment of the present invention, the composition for collecting placental cells contains the specified inhibitor of apoptosis and the specified perfluorocarbons, able to carry oxygen in the emulsion. In another embodiment of the present invention, the composition for collecting placental cells are additionally comprises an emulsifier, in particular letit is N. In another embodiment, the present invention during contact with cells of the placenta temperature specified inhibitor of apoptosis and specified perfluorocarbons ranges from approximately 0°to approximately 25°C. In another more specific embodiment, the present invention during contact with cells of the placenta temperature specified inhibitor of apoptosis and specified perfluorocarbons is from about 2°to about 10°C., or from about 2°to about 5°C. In another more specific embodiment, the present invention specified the contacting is carried out in the process of transporting a specified population of stem cells. In another more specific embodiment, the present invention specified the contacting is carried out in the process of freezing and thawing specified population of stem cells.

In another embodiment, the present invention placental perfusion solution and/or cells of the placental perfusion solution can collect and maintain by contacting perfusion solution and/or cells with an inhibitor of apoptosis and organochromium connection, with the indicated inhibitor of apoptosis is present in an amount and for a time sufficient to reduce or prevent apoptosis of cells, with whom avanyu with the perfusion solution or placental cells, which is not contacted with the inhibitor of apoptosis.

In a specific embodiment, the present invention organochromium connection is the UW solution (described in U.S. patent No. 4798824; also known as VIASPAN™; see also Southard et al.,Transplantation49(2):251-257 (1990) or the solution described in issued in the name of Stern et al. U.S. patent No. 5552267. In another embodiment, the present invention organ composition represents gidroxiatilkrahmal, lactobionic acid, raffinose, or combinations thereof. In another embodiment of the present invention, the composition for collecting placental cells are additionally includes perfluorocarbons, able to carry oxygen in the form of a two-phase system, and in the form of an emulsion.

In another embodiment of the method according to the present invention placental perfusion solution or cells of the placenta in contact with the composition for collecting placental cells, which is in the process of perfusion includes an inhibitor of apoptosis and perfluorocarbons, able to carry oxygen, organ-preserving compound, or a combination of both. In another embodiment of the present invention the cells of the placenta in contact with the specified connection to collect stem cells once you have collected by perfusion.

As a rule, in the process of collection, enrichment and selection of placental cells, p is edocfile, minimize or eliminate the stress caused by hypoxia, and mechanical stress. Therefore, in another embodiment of the method according to the present invention placental perfusion solution, the cells of the placenta or population of placental cells during collection, enrichment or selection are subjected to conditions of hypoxia within less than six hours during the specified retention period, under the condition of hypoxia is that the oxygen concentration is less than the normal concentration of oxygen in the blood. In a more specific embodiment of the present invention indicated a population of placental cells is subjected to the specified conditions of hypoxia within less than two hours during the specified retention period. In another more specific embodiment, the present invention indicated a population of placental cells during collection, enrichment or selection is subjected to the specified conditions of hypoxia within less than one hour, or less than thirty minutes, or not subject to conditions of hypoxia. In another specific embodiment, the present invention indicated a population of placental cells not subjected to shear stress during collection, enrichment or selection.

Proposed in the present invention the cells of the placenta can be saved in terms of biotemperature, for example, in cf the de storage conditions biotemperature in small containers, in particular ampoules. Suitable environment for storing in conditions of biotemperature includes, but not limited to, the culture medium, which contains, in particular, a growing medium, or medium for freezing, for example, commercially available medium for freezing, in particular, C2695, C2639 or C6039 (Sigma). The storage medium in terms of biotemperature preferably includes DMSO (dimethyl sulfoxide) concentration, in particular, approximately 10% (vol./vol.). The storage medium in terms of biotemperature may contain additional agents, such as methylcellulose and/or glycol. In the process of saving in terms of biotemperature cells of the placenta, preferably cooled at a rate of about 1°C/min primary biotemperature approximately from -80°C. to about -180°C., preferably, from about -125°C. to about -140°C.) Cooled to biotemperature cells of the placenta can be transferred in liquid nitrogen and stored until thawing before use. In some embodiments, implementation of the present invention, for example, once the ampoule has reached a temperature of about -90°C, transferred into a store for storing in liquid nitrogen. Stored cells, mostly thawed at a temperature of from about 25°to about 40°C., typically the military, at a temperature of approximately 37°C.

5.7. The use of placental perfusion solution, PINK cells, combined natural killer cells of the placenta to inhibit the growth of tumor cells

In the present invention are also methods of inhibition of growth, in particular, proliferation, tumor cells using placental perfusion solution, cells isolated from placental perfusion solution, the selected combined natural killer cells or selected natural killer cells of the placenta, in particular, selected from the placenta helper and natural killer cells.

In accordance with one embodiments of the present invention, it is proposed a method of suppressing the proliferation of tumor cells or a variety of tumor cells which comprises contacting the tumor cells or a variety of tumor cells with placental perfusion solution, the cells of placental perfusion solution, the selected combined natural killer cells and/or PINK cells, such that proliferation of tumor cells or a variety of tumor cells is markedly reduced, compared with a tumor cell or many tumor cells of the same type that is not contacted with placental perfusion solution, the cells of placental perfusion solution, allocated combined the natural killer cells and/or PINK cells.

In the present description “contacts” in one embodiment, the present invention covers direct physical, for example, cell-cell contact between placental perfusion solution, the cells of placental perfusion solution, natural killer cells of the placenta, in particular, supporting the natural killer cells of the placenta, and/or selected and combined natural killer cells; and a tumor cell or a variety of tumor cells. In another embodiment of the present invention “contacting” encompasses the same physical environment, in particular, placental perfusion solution, the cells of placental perfusion solution, natural killer cells of the placenta, in particular, supporting the natural killer cells of the placenta and/or the selected combined natural killer cells are placed in the same container (in particular, in a Cup with culture, advance the tablet), and a tumor cell or a variety of tumor cells. In another embodiment of the present invention “contacts” placental perfusion solution, cells placental perfusion solution, combined natural killer cells or supporting natural killer cells of the placenta and malignant tumor or a variety of tumor cells is carried out, in particular, the ay injection or infusion placental perfusion solution or cells, in particular, cells of placental perfusion solution, combined natural killer cells or supporting natural killer cells of the placenta, an individual, for example, a person who has a tumor cell or a variety of tumor cells, for example cancer patient.

In some embodiments, implementation of the present invention placental perfusion solution used in any quantity, which leads to a detectable therapeutic effect for the individual who has a tumor cell or a variety of tumor cells, for example, for a cancer patient. In some other embodiments, implementation of the present invention the cells of the placental perfusion solution, supporting natural killer cells of the placenta and/or combined natural killer cells are used in any quantity, which leads to a detectable therapeutic effect for the individual who has a tumor cell or a variety of tumor cells. Thus, in another embodiment, the present invention proposes a method of suppressing the proliferation of tumor cells or a variety of tumor cells which comprises contacting the tumor cells or a variety of tumor cells with placental perfusion solution, the cells of placental perfusion solution and/or isolated from the placenta helper is naturalnie killer cells, and/or lots of PINK cells within an individual so that this contact brings therapeutic effect of a given individual that can detect or confirm.

In the present description “therapeutic effect” includes, but not limited to, reduction in tumor size; reduction or cessation of tumor growth; decrease in the number of cancer cells in the tissue sample, in particular in a blood sample, per unit volume; clinical improvement in any symptom specific type of cancer, which is present in the individual; reduction or termination of a worsening of any symptom specific type of cancer that has a individual, and so on Contacting the placental perfusion solution, cells placental perfusion solution and/or PINK cells, which leads to one or more similar therapeutic effects, considered to be therapeutically effective.

In some embodiments, implementation of the present invention the cells of the placental perfusion solution, in particular, nucleated cells from placental perfusion solution, combined natural killer cells, and/or support natural killer cells of the placenta used in any quantity or number that leads to a detectable therapeutic effect for the individual who has a tumor cell or a variety of tumor cells, in which lastnosti cancer patient. Cells placental perfusion solution, combined natural killer cells and/or natural killer cells of the placenta, in particular, supporting the natural killer cells of the placenta, you can enter a specified individual as the number of cells, in particular, a given individual can be assigned approximately at least about, or no more than about 1×105, 5×105, 1×106, 5×106, 1×107, 5×107, 1×108, 5×108, 1×109, 5×109, 1×1010or 5×1010cells placental perfusion solution, combined natural killer cells and/or natural killer cells of the placenta. In other embodiments, implementation of the present invention the cells of the placental perfusion solution, combined natural killer cells and/or isolated from the placenta helper and natural killer cells of the placenta, you can enter such individual in the form of a certain number of cells, in particular, a given individual can be assigned approximately at least about, or no more than about 1×105, 5×105, 1×106, 5×106, 1×107, 5×107, 1×108, 5×108, 1×109, 5×109, 1×1010or 5×1010cells placental perfusion solution, combined natural killer cells and/or natural cells is of Ellerbe per kilogram of weight of the individual. Cells placental perfusion solution and/or isolated from the placenta helper and natural killer cells of the placenta, you can enter such individual in accordance with an approximate relation between the cells of the placental perfusion solution and/or natural killer cells of the placenta, in particular, supporting the natural killer cells of the placenta and tumor cells of a specified individual. For example, cells of placental perfusion solution and/or natural killer cells of the placenta, in particular, supporting the natural killer cells of the placenta, you can enter a specified individual in relation to approximately at least about, or no more than about 1:1, 1:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 15:1, 20:1, 25:1, 30:1, 35:1, 40:1, 45:1, 50:1, 55:1, 60:1, 65:1, 70:1, 75:1, 80:1, 85:1, 90:1, 95:1 or 100:1 to the number of tumor cells of a specified individual. The number of tumor cells in such individuals can be assessed, in particular, by counting the number of tumor cells taken from the individual tissue sample, for example

blood, biopsy, etc., In specific embodiments, the implementation of the present invention, in particular, for solid tumors, this calculation is performed in conjunction with visualization of the tumor or tumors in order to determine the approximate volume of the tumor.

In addition, the present invention proposes a method of suppression is roliferation tumor cells or a variety of tumor cells using combinations of placental perfusion solution, cells placental perfusion solution, combined natural killer cells, and/or support of natural killer cells isolated from placenta. In various embodiments, implementation of the present invention proposes a method of suppressing the proliferation of tumor cells or a variety of tumor cells which comprises contacting the indicated tumor cells or the indicated tumor cells with placental perfusion solution, supplemented by a variety of cells placental perfusion solution or PINK cells; cells of placental perfusion solution, supplemented with placental perfusion solution or a lot of PINK cells; PINK cells supplemented with placental perfusion solution and the cells of the placental perfusion solution, lots of PINK cells and many combined natural killer cells; with many combined natural killer cells, and many cells of placental perfusion solution; with placental perfusion solution, supplemented with combined natural killer cells; or a combination of all of these placental perfusion solution, cells placental perfusion solution, combined natural killer cells, and PINK cells.

For example, in a specific embodiment, the present invention proliferation of tumor cells or a variety of tumor cells is inhibited placental perfusion solution, the l is United by many cells of placental perfusion solution, combined natural killer cells, and/or other natural killer cells of the placenta. For example, in specific embodiments, the implementation of the present invention each ml of placental perfusion solution supplemented with about 1×104, 5×104, 1×105, 5×105, 1×106, 5×106or more cells of placental perfusion solution or support natural killer cells of the placenta. In other specific embodiments, the implementation of the present invention placental perfusion solution, in particular, one unit (i.e., the collection from one placenta), or approximately 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950 or 1000 ml of perfusion solution complement of approximately 1×104, 5×104, 1×105, 5×105, 1×106, 5×106, 1×107, 5×107, 1×108, 5×108or more PINK cells, combined killer cells and/or cells of placental perfusion solution per milliliter, or 1×104, 5×104, 1×105, 5×105, 1×106, 5×106, 1×107, 5×107, 1×108, 5×108, 1×109, 5×109, 1×1010, 5×1010, 1×1011or more PINK cells, combined killer cells and/or cells of placental perfusion solution.

In another specific embodiment, the present invention proliferation of tumor CL the TCA or a variety of tumor cells is suppressed by many cells of placental perfusion solution, augmented placental perfusion solution, the combined natural killer cells, and/or support natural killer cells of the placenta. In more specific embodiments, the implementation of the present invention is approximately 1×104, 5×104, 1×105, 5×105, 1×106, 5×106, 1×107, 5×107, 1×108, 5×108or more cells of placental perfusion solution per milliliter, or 1×104, 5×104, 1×105, 5×105, 1×106, 5×106, 1×107, 5×107, 1×108, 5×108, 1×109, 5×109, 1×1010, 5×10, 1×1011or more cells of placental perfusion solution complement about or at least about 1×104, 5×104, 1×105, 5×105, 1×106, 5×106, 1×107, 5×107, 1×108, 5×108or more PINK cells and/or combined natural killer cells per milliliter, or 1×104, 5×104, 1×105, 5×105, 1×106, 5×106, 1×107, 5×107, 1×108, 5×108, 1×109, 5×109, 1×1010, 5×1010, 1×1011or more PINK cells. In other more specific embodiments of implementation of the present invention is approximately 1×104, 5×104, 1×105, 5×105, 1×106, 5×106, 1×107, 5×107, 1×108, 5×108or more of the notches placental perfusion solution, PINK cells and/or combined natural killer cells per milliliter, or 1×104, 5×104, 1×105, 5×105, 1×106, 5×106, 1×107, 5×107, 1×108, 5×108, 1×109, 5×109, 1×1010, 5×1010, 1×1011or more cells of placental perfusion solution supplemented with about or at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950 or 1000 ml of perfusion solution or about 1 unit of perfusion solution.

In another specific embodiment, the present invention proliferation of tumor cells or a variety of tumor cells is suppressed by many of the supporting natural killer cells of the placenta, augmented placental perfusion solution, the cells of placental perfusion solution and/or combined natural killer cells. In more specific embodiments, the implementation of the present invention is approximately 1×104, 5×104, 1×105, 5×105, 1×106, 5×106, 1×107, 5×107, 1×108, 5×108or more auxiliary natural killer cells of the placenta, or 1×104, 5×104, 1×105, 5×105, 1×106, 5×106, 1× 107, 5×107, 1×108, 5×108, 1×109, 5×109, 1×1010, 5×10, 1×1011 4, 5×104, 1×105, 5×105, 1×106, 5×106, 1×107, 5×107, 1×108, 5×108or more cells of placental perfusion solution and/or combined natural killer cells per milliliter, or 1×104, 5×104, 1×105, 5×105, 1×106, 5×106, 1×107, 5×107, 1×108, 5×108, 1×109, 5×109, 1×1010, 5×1010, 1×1011or more cells of placental perfusion solution and/or combined natural killer cells. In other more specific embodiments of implementation of the present invention is approximately 1×104, 5×104, 1×105, 5×105, 1×106, 5×106, 1×107, 5×107, 1×108, 5×108or more cells of placental perfusion solution and/or combined natural killer cells per milliliter, or 1×104, 5×104, 1×105, 5×105, 1×106, 5×106, 1×107, 5×107, 1×108, 5×108, 1×109, 5×109, 1×1010, 5×1010, 1×1011or more helper cells, natural killer cells of the placenta and/or combined natural killer cells, complement with the help of about or at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950 or 1000 ml perfusate approximately 1 unit of perfusion solution.

In another embodiment, the present invention proliferation of tumor cells or a variety of tumor cells is suppressed by contacting the tumor cells or tumor cells with placental perfusion solution, the cells of placental perfusion solution, PINK cells and/or combined natural killer cells, supplemented adherent stem cells of the placenta. In specific embodiments, the implementation of the present invention placental perfusion solution, the cells of placental perfusion solution or PINK cells complement of approximately 1×104, 5×104, 1×105, 5×105, 1×106, 5×106, 1×107, 5×107, 1×108, 5×108or more adherent stem placental cells per milliliter, or 1×104, 5×104, 1×105, 5×105, 1×106, 5×106, 1×107, 5×107, 1×108, 5×108, 1×109, 5×109, 1×1010, 5×1010, 1×1011or more adherent stem cells of the placenta.

In another embodiment, the present invention proliferation of tumor cells or a variety of tumor cells is suppressed by contacting the tumor cells or tumor cells with placental perfusion solution, the cells of placental perfusion solution, the combined natural killer cells and/or PINK cells, augmented environments is th, which is air-conditioned adherent stem cells of the placenta, in particular, augmented 0,1; 0,2; 0,3; 0,4; 0,5; 0,6; 0,1; 0,8; 0,9; 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 ml of culture medium, which is air-conditioned adherent stem cells of the placenta, per unit of perfusion solution, cell perfusion solution, combined natural killer cells and/or PINK cells, or 104, 105, 106, 107, 108, 109or 1010cells.

In other embodiments, implementation of the present invention placental perfusion solution, the cells of placental perfusion solution, the natural killer cells of the placenta, in particular PINK cells, combined natural killer cells, or combinations thereof, and pooled funds are used in the form in which they originally received, i.e., the perfusion solution in the form in which he obtained during perfusion, cells placental perfusion solution in the form in which they are isolated from such perfusion solution, combined natural killer cells of similar perfusion solution and a compatible cord blood, or PINK cells, isolated from similar perfusion solution or similar cells placental perfusion solution. In other embodiments, implementation of the present invention placental perfusion solution, the cells of placental perfusion solution, PINK cells, and combinations thereof and pooled funds processed before use. For example, the placental perfusion solution which can be used in its raw, not exposed to processing the form in which he isolated from the placenta. Placental perfusion solution may also be subjected before use processing, for example, by negative selection of one or more cell types; by reducing the volume through dehydration; by lyophilization or re-hydration, etc. Similarly, a population of cells perfusion solution can be used in the originally isolated from placental perfusion solution form, in particular in the form of a set of nucleated cells from placental perfusion solution, or may be processed, for example, to remove one or more types of cells (including erythrocytes). PINK cells can be used in the originally isolated from placental perfusion solution form, in particular, when using the beads CD56, or can be processed, for example, to remove one or more types of cells other than killer cells.

In another embodiment, the present invention proposes a method of suppressing the proliferation of tumor cells or neoplastic cells, which consists in contacting the tumor cells or tumor cells with placental perfusion solution, the cells of placental perfusion solution, PINK cells, combined natural killer cells and their combined funds or to what minutiae, while specified placental perfusion solution, the cells of placental perfusion solution, PINK cells, combined natural killer cells or pooled funds, or a combination had contact with interleukin-2 (IL-2) within a certain period of time before the specified contacts. In certain embodiments of the implementation of the present invention specified period of time is about, is at least about or less than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46 or 48 before the specified contact.

The perfusion solution, the cells of placental perfusion solution, PINK cells, combined natural killer cells or pooled funds and/or combinations can be assigned to an individual who has cancer, one time, or assign them to an individual who has cancer, during anti-cancer therapy, or be assigned several times, in particular once in each 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22 or 23 hours, or once in every 1, 2, 3, 4, 5, 6 or 7 day, or once in every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more a week during treatment. The perfusion solution, the cells of placental perfusion solution, PINK cells, pooled funds and/or combinations can be entered without regard to whether appointed if the person who has cancer, or the person with the tumor cells, the perfusion solution, the cells and placental perfusion solution, PINK cells, pooled funds and/or combinations in the past. Thus, the methods proposed in the present invention comprise administration to a person who has cancer, or the person with the tumor cells, any combination of placental perfusion solution, cells placental perfusion solution, PINK cells, pooled funds and/or combinations.

In a specific embodiment of the present invention, tumor cells are the blood cells of the tumor. In various specific embodiments, implementation of the present invention, tumor cells are cells of primary carcinoma of the epithelial duct cells, leukemia cells, acute T-cell leukemia cells, chronic myeloid lymphoma (CML) cells, acute myelogenous leukemia cells, chronic myelogenous leukemia (CML) cells, lung carcinoma cells, adenocarcinoma of the colon, cells histiocytomas lymphoma cells, multiple myeloma cells, retinoblastoma cells, colorectal carcinoma or cells of colorectal adenocarcinoma.

Placental perfusion solution, the cells of placental perfusion solution, PINK cells, combined natural killer cells, pooled funds and/or combinations may be part of a treatment regimen for cancer, which includes one or more other anti-cancer remedies. Such anticancer good is known from the technical field. Specific anti-cancer drugs, which can be assigned to an individual having cancer, in addition to the perfusion solution, the cells of the perfusion solution, PINK cells, their combined funds and/or combinations are, but not limited to: acivicin; aclarubicin; acetasol hydrochloride; Acronis; adozelesin; aldesleukin; altretamin; ambomycin; ametantrone acetate; amsacrine; anastrozole; astromicin; asparaginase; aspirin; azacytidine; asettaa; azotomycin; batimastat; benzodepa; bikalutamid; bisantrene hydrochloride; bisnafide dimesylate; bizelesin; bleomycin sulfate; brequinar sodium; bropirimine; busulfan; Antinomian; calusterone; caracemide; carbetimer; carboplatin; carmustine; karubitsin hydrochloride; carzelesin; Cedeira; celecoxib (a COX-2 inhibitor); chlorambucil; cirolemycin; cisplatin; cladribine; Kristol mesilate; cyclophosphamide; cytarabine; dacarbazine; dactinomycin; daunorubicin hydrochloride; decitabine; decompletion; deazaguanine; deazaguanine mesilate; diazinon; docetaxel; doxorubicin; doxorubicin hydrochloride; droloxifene; droloxifene citrate; dromostanolone propionate; deatomizer; edatrexate; avlonitis hydrochloride; elsamitrucin; anoplate; enpromate; epirubicin; epirubicin hydrochloride; arbolada; zorubicin hydrochloride; estramustine; estramustine sodium phosphate; etanidazole; etoposide; etoposide phosphate; atopen; fad is ozol hydrochloride; fazarabine; phenetidine; floxuridine; fludarabine phosphate; fluorouracil; ferritin; fashion; fostriecin sodium; gemcitabine; gemcitabine hydrochloride; hydroxyurea; idarubitsin hydrochloride; ifosfamide; ilmofosine; iproplatin; irinotecan; irinotecan hydrochloride; lanreotide acetate; letrozole; leuprolide acetate; lioresal hydrochloride; lometrexol sodium; lomustin; losoxantrone hydrochloride; masoprocol; maytansine; mechlorethamine hydrochloride; megestrol acetate; melengestrol acetate; melphalan; menogaril; mercaptopurine; methotrexate; methotrexate sodium; metapan; maturated; matindale; microcin; mitotropin; mitogillin; mitomycin; mitomycin; mitosis; mitotane; mitoxantrone hydrochloride; mycofenolate acid; nocodazole; nogalamycin; ormaplatin; oxysure; paclitaxel; pegaspargase; polymycin; pentamycin; peplomycin sulfate; perforated; pipobroman; piposulfan; piroxantrone hydrochloride; plicamycin; plomelin; porfimer sodium; porfiromycin; prednimustine; procarbazine hydrochloride; puromycin; puromycin hydrochloride; pyrazofurin; ibuprin; safingol; safingol hydrochloride; semustine; contrast; spartacat sodium; sparsomycin; spirogermanium hydrochloride; spiramycin; spiroplatin; streptonigrin; streptozocin; alienor; talisayan; tecogen sodium; Taxotere; tegafur; Alexandre hydrochloride; ]; tenpo the ID; teraxion; testolactone; timipre; tioguanin; thiotepa; teatterin; tirapazamine; toremifene citrate; trestolone acetate; triciribine phosphate; trimetrexate; trimetrexate the glucuronate; triptorelin; tubulosa hydrochloride; uracil mustard; uredepa; vapreotide; verteporfin; vinblastine sulfate; vincristine sulfate; vindesine; vindesine sulfate; benefitin sulfate; Inglesina sulfate; winnersin sulfate; vinorelbine tartrate; vinosity sulfate; ventolin sulfate; vorozole; senility; zinostatin; and zorubicin hydrochloride.

Other anti-cancer tools are, but not limited to: 20-EPI-1,25-hydroxyvitamin D3; 5-itinerarary; abiraterone; aclarubicin; allfusion; deciphered; adozelesin; aldesleukin; antagonists ALL-TK; altretamin; ambamustine; amidax; amifostine; aminolevulinic acid; amrubicin; amsacrine; anagrelide; anastrozole; Andrographolide; angiogenesis inhibitors; antagonist D; antagonist G; entrelacs; antidotale expressed morphogenetic protein-1; antiandrogen, carcinoma of the prostate; antiestrogen; antineoplaston; antisense oligonucleotides; aphidicolin glycinate; gene modulators of apoptosis; regulators of apoptosis; Euronova acid; ara-CDP-DL-PTBA; argininemia; isolagen; atamestane; attemptin; achinstein 1; achinstein 2; achinstein 3; azasetron; anatoxin; asteroid; made in the derivative baccatin III; balana; batimastat; antagonists BCR/ABL; benzocaine; benzoyltartaric; derivatives of beta-lactam beta-alamin; butaclamol B; betulin acid; an inhibitor of bFGF; bikalutamid; bisantrene; besuseradminclient; bisnafide; bitraten A; bizelesin; Brevet; bropirimine; budotitane; buthionine sulfoximine; calcipotriol; calphostin C; derivatives camptothecin; capecitabine; carboxamid-aminotriazol; carboxamidates; CaRest M3; CARN 700; inhibitor of cartilage; carzelesin; inhibitors caseinline (ICOS); castanospermine; cecropin B; cetrorelix; chlorins; chloroquinoxalin the sulfonamide; cicaprost; CIS-porphyrin; cladribine; analogues clomiphene; clotrimazole; colimycin a; colimycin B; combretastatin A4; analogues of combretastatin; convenin; kambezidis 816; Kristol; cryptophycin 8; derivatives cryptophycin; curacin a; cyclopentadecanone; cyclopean; cephamycin; cytarabine ocfosfate; cytolytic factor; cytostatin; daclizumab; decitabine; dehydrodidemnin B; deslorelin; dexamethasone; Taxifolin; dexrazoxane; dexverapamil; diazinon; didemnin B; detox; diethylnitrosamine; dihydro-5-azacytidine; 9-dihydroxy; dioxazine; diphenylpyraline; docetaxel; docosanol; dolasetron; doxifluridine; doxorubicin; droloxifene; dronabinol; duocarmycin SA; ebselen; elastin; edelfosine; edrecolomab; eflornithine; elements; Amy is EUR; epirubicin; epristeride; analogues of estramustine; agonists of estrogen; estrogen antagonists; etanidazole; etoposide phosphate; exemestane; fadrozole; fazarabine; phenetidine; filgrastim; finasteride; flavopiridol; fileselection; fluasterone; fludarabine; forceunauthorized hydrochloride; forenames; formestane; fostriecin; fotemustine; texaphyrin gadolinium; gallium nitrate; Galitsin; ganirelix; inhibitors gelatinase; gemcitabine; inhibitors of glutathione; HaSulam; heregulin; gesammelt biocetamol; hypericin; ibandronic acid; idarubitsin; idoxifene; Idamante; ilmofosine; ilomastat; imatinib (in particular, GLEEVEC®), imiqimod; peptides possessing immunostimulatory activity; inhibitor of receptor insulin-like growth factor-1; interferon agonists; interferons; interleukins; iobenguane; iododeoxyuridine; ipomeanol-4; ireplace; irsogladine; isomerases; solomakhin B; fusetron; jasplakinolide; kahalalide F; lamellarin-N triacetate; lanreotide; lanemile; lenograstim; lentinan sulfate; leptostachys; letrozole; inhibitory factor leukemia; alpha-interferon from leukocytes; leuprolide + estrogen + progesterone; leiprorelina; levamisole; lioresal; similar linear polyamine; lipophilic disaccharide glycosides peptide; lipophilic platinum compounds; lissoclinum 7; lobaplatin; lubricin; lometrexol; lonidamine; lasok the Andron; doxorubin; lurtotecan; texaphyrin lutetium; lisofylline; lytic peptides; maytansine; sandostatin a; marimastat; masoprocol; maspin; inhibitors matrilysin; inhibitors of matrix metalloproteinases; menogaril; merbanan; peterlin; methionine; metoclopramide; inhibitor of MIF; mifepristone; miltefosine; Miramistin; mitoguazone; mitolactol; analogues of mitomycin; mitonafide; mycotoxin-fibroblast growth factor-saporin; mitoxantrone; operatin; molgramostim; Erbitux, human chorionic gonadotropin; monophosphorylated A + streptokinase cell wall of mycobacteria; mopidamol; anticancer agent mustard; megaproxy B; extract cell wall of mycobacteria; mylapore; N-azetidinone; N-substituted benzamide; nafarelin; Agresti; naloxone + pentazocine; nipawin; Natterer; nartograstim; nedaplatin; nemorubicin; pridanova acid; nilutamide; nizamettin; modulators of nitric oxide; nitroxide antioxidant; nitrolon; oblimersen (GENASENSE®); O6-benzylguanine; octreotide; okizeme; oligonucleotides; onapristone; ondansetron; oracin; oral inducing factor cytokines; ormaplatin; asteron; oxaliplatin; axiomized; paclitaxel; analogues of paclitaxel; derivatives paclitaxel; palyulin; palmitoylation; panikanova acid; panaxytriol; promife; pyrabactin; pallidin; pegaspargase is; peltatin; pentosanpolysulfate sodium; pentostatin; petrosal; perflubron; perforated; parallelly alcohol; fansinating; phenylacetate; inhibitors of phosphatase; picibanil; pilocarpine hydrochloride; pirarubicin; piritrexim; placedin a; placein B; inhibitor of plasminogen activator; a complex of platinum; platinum compounds; platinum-criminaly complex; porfimer sodium; porfiromycin; prednisone; propyl-bis-acridan; prostaglandin J2; proteasome inhibitors; immunomodulator on the basis of protein A, an inhibitor of protein kinase C, an inhibitor of protein kinase C, microalgae; inhibitors of protein-tyrosine phosphatase; inhibitors of the purine nucleoside phosphorylase; the purpurin; pyrazoloacridine; conjugate pyridoxamine hemoglobin and polyoxyethylene; antagonists raf; raltitrexed; ramosetron; inhibitors of ras farnesyl-protein transferase; ras inhibitors; inhibitors of ras-GAP; demetilirovanny reality; etidronate rhenium Re 186; rhizoxin; ribozymes; RII retinamide; rohitukine; romantic; roquinimex; rubiginosa B1; robaxin; safingol; sintobin; SarCNU; sarcophyton A; sargramostim; mimetics Sdi 1; semustine; inhibitor of cell aging 1; sense oligonucleotides; inhibitors of signal transmission; sizofiran; sobuzoxane; borocaptate sodium; sodium phenylacetate; solvera; somatomedin-binding protein; sonarmen; spartaaa acid; spicamycin D; spiramycin; plenipoten; spongistatin 1; qualmin; stipend; inhibitors stromelysin; solifenacin; antagonist super active vasoactive putting peptide; coralista; suramin; swainsonine; tallimustine; tamoxifen methiodide; terramycin; tazarotene; tecogen sodium; Ghafur; tolerability; telomerase inhibitors; ]; teniposide; tetrachlorodecaoxide; tetrasomy; teleblaster; thiocoraline; thrombopoietin; mimetic of thrombopoetin; thymalfasin; agonist of the receptor of thymopoietin; timorian; thyroidstimulating hormone; tin ethyl-adipocere; tirapazamine; titanocene bichloride; topsentin; toremifene; inhibitors broadcast; tretinoin; triacetyluridine; triciribine; trimetrexate; triptorelin; tropisetron; turosteride; tyrosine kinase inhibitors; tyrphostin; inhibitors UBC; ubenimex; inhibiting the growth factor from the urogenital sinus; antagonists of the receptor for urokinase; vapreotide; variolin B; valarezo; vermin; verdini; verteporfin; vinorelbine; Wincanton; vitaxin; vorozole; sonotron; senility; salaskar; and zinostatin stimulater.

5.8. Processing of natural killer cells immunomodulatory compounds

Selected natural killer cells, in particular PINK cells or combined natural killer cells, considered in the present description, can be subjected to processing immunomodulatory joint is m, for example to enter into contact with the immunomodulatory compound, in order to enhance the antitumor activity of these cells. Thus, the present invention proposes a method of increasing the cytotoxicity of natural killer cells against tumor cell which comprises contacting the natural cell-killer with an immunomodulatory compound over time and with concentration, sufficient to natural cell-killer showed enhanced cytotoxicity against tumor cell compared to a natural cell-killer, which is not contacted with the immunomodulatory compound. In accordance with another embodiment of the present invention proposes a method of enhancing expression of granzyme In natural cells killer cells, which comprises contacting the natural cell-killer with an immunomodulatory compound over time and with concentration, sufficient to natural cell-killer showed increased expression of granzyme In comparison with natural cell-killer, which is not contacted with the immunomodulatory compound. Immunomodulatory compound can be any below the connection, in particular, lenalidomide or pomalidomide.

In the present invention offer the W is also a way of increasing the cytotoxicity of the population of natural killer cells, in particular PINK cells or combined natural killer cells, against many tumor cells, which comprises contacting the population of natural killer cells with an immunomodulatory compound over time and with concentration, sufficient for the population of natural killer cells demonstrated detektiruya increased cytotoxicity with respect to a given set of tumor cells, compared with an equivalent number of natural killer cells that are not contacted with the immunomodulatory compound. In accordance with another embodiment of the present invention proposes a method of enhancing expression of granzyme In the population of natural killer cells, which comprises contacting the population of natural killer cells with an immunomodulatory compound over time and with concentration, sufficient for the population of natural killer cells expressed detective increased amount of granzyme In, compared with an equivalent number of natural killer cells that are not contacted with the immunomodulatory compound. In a specific embodiment of the present invention, a specified population of natural killer cells contained together with the cells of placental perfusion solution,in particular with the total number of nucleated cells, isolated from placental perfusion solution.

In specific embodiments, the embodiments of the above embodiments of the present invention natural killer cells are CD56+, CD16‾, auxiliary natural killer cells of the placenta (PINK cells). In another particular variant embodiment of the above embodiments of the present invention natural killer cells are combined natural killer cells, i.e., natural killer cells from compatible with each other placental perfusion solution and umbilical cord blood.

In another specific embodiment, the present invention specified variety of natural killer cells, in particular, PINK cells or combined natural killer cells, which are contacted with the specified immunomodulatory compound, expresses one or more of BAX, CCL5, CCR5, CSF2, FAS, GUSB, IL2RA or TNFRSF18 at a higher level than an equivalent number of specified natural killer cells that are not contacted with the specified immunomodulatory compound. In another specific embodiment, the present invention specified variety of natural killer cells, in particular PINK cells that were in contact with the specified immunomodulatory compound, Express one or more of the ACT, BAX, CCL2, CCL3, CCL5, CCR5, CSF1, CSF2, ECE1, FAS, GNLY, GUSB, GZMB, IL1A, IL2RA, IL8, IL10, LTA, PRF1, PTGS2, SKI and TBX21 at a higher level than an equivalent number of specified natural killer cells that are not contacted with the specified immunomodulatory compound.

In the present invention it is also proposed a method of increasing the cytotoxicity of the cell population of placental perfusion solution and person, in particular together nucleated cells from placental perfusion solution, against many tumor cells, which comprises contacting cells placental perfusion solution with an immunomodulatory compound over time and with concentration, sufficient for the cells of placental perfusion solution showed detektiruya increased cytotoxicity with respect to a given set of tumor cells, compared with an equivalent number of cells placental perfusion solution, which is not contacted with the immunomodulatory compound. In accordance with another embodiment of the present invention proposes a method of enhancing expression of granzyme In the population of cells of placental perfusion solution, which comprises contacting the population of cells of placental perfusion solution with an immunomodulatory compound over time and with concentration, sufficient for a population of placental cells p is hwseta expressed detective increased amount of granzyme, compared with an equivalent number of cells placental perfusion solution, which is not contacted with the immunomodulatory compound.

Immunomodulatory compounds or can be purchased commercially or can be obtained in accordance with the methods described in the patents or patent publications cited in the present description and which are all included in the present description by reference. In addition, optically clear compositions can be obtained by asymmetric synthesis or cleavage using known disintegrating agents or chiral columns, and other standard methods of synthetic organic chemistry. Immunomodulatory compounds may be racemic, stereochemical enriched or pure stereochemical and can cover their pharmaceutically acceptable salt, solvate and prodrug.

In the context of the present description, unless otherwise specified, the terms “immunomodulatory compounds comprise organic molecules of small size, which significantly inhibit TNF-α, LPS-induced monocytes IL-1β and IL-12 and partially inhibit the production of IL-6. Specific examples of immunomodulatory compounds are lenalidomide, pomalidomide or thalidomide.

Specific examples of immunomodulatory compounds R is t, but not limited to, cyano - or carboxyesterase derivatives substituted styrene, such as disclosed in U.S. patent No. 5929117; 1-oxo-2-(2,6-dioxo-3-foreperiod-3-yl)isoindolines and 1,3-dioxo-2-(2,6-dioxo-3-foreperiod-3-yl)isoindoline, such as disclosed in U.S. patent No. 5874448 and 5955476; Tetra-substituted 2-(2,6-dioxopiperidin-3-yl)-1-occaisonally described in U.S. patent No. 5798368; 1-oxo and 1,3-2-(2,6-dioxopiperidin-3-yl)isoindoline (in particular, 4-methylamine derivative of thalidomide), including, but not limited to, derivatives such as disclosed in U.S. patent No. 5635517, 6476052, 6555554, and 6403613; 1-oxo and 1,3-dioxoindoline, zameshannye 4 - or 5-position indolinone cycle (in particular, 4-(4-amino-1,3-doxasozin-2-yl)-4-carbarnoyl-butane acid)described in U.S. patent No. 6380239; isoindoline-1-he and isoindoline-1,3-dione substituted in the 2-position of 2,6-dioxo-3-hydroxypiperidine-5-yl (in particular, 2-(2,6-dioxo-3-hydroxy-5-foreperiod-5-yl)-4-iminoisoindolin-1-he), is shown in U.S. patent No. 6458810; class polypeptide cyclic amides disclosed in U.S. patent No. 5698579 and 5877200; aminosilicone, as well as analogues, the hydrolysis products, metabolites, derivatives and precursors aminosilicone and substituted 2-(2,6-dioxopiperidin-3-yl)phthalimide and substituted 2-(2,6-dioxopiperidin-3-yl)-1-occaisonaly, such as disclosed in U.S. Pat is ntah U.S. No. 6281230 and 6316471; and isoindoline compounds such as described in patent publication U.S. No. 2003/0045552 A1, U.S. patent No. 7091353 and international application WO 02/059106. The content of each of the above in the present description of patents and patent publications are included in this description by reference. Immunomodulatory compounds do not include thalidomide.

In specific embodiments, the implementation of the present invention immunomodulatory compounds are 1-oxo and 1,3-dioxo-2-(2,6-dioxopiperidin-3-yl)isoindoline, substituted amino group in the benzene cycle, as described in U.S. patent No. 5635517, which is entirely included in the present description by reference. These compounds have the structure I

where one of X and Y represents C=O, and the other of X and Y represents C=O or CH2and R2denotes a hydrogen atom or lower alkyl, in particular methyl. Specific immunomodulatory compounds include, but are not limited to:

1-oxo-2-(2,6-dioxopiperidin-3-yl)-4-iminoisoindolin;

1-oxo-2-(2,6-dioxopiperidin-3-yl)-5-iminoisoindolin;

1-oxo-2-(2,6-dioxopiperidin-3-yl)-6-iminoisoindolin;

1-oxo-2-(2,6-dioxopiperidin-3-yl)-7-iminoisoindolin;

1,3-dioxo-2-(2,6-dioxopiperidin-3-yl)-4-iminoisoindolin; and

1,3-dioxo-2-(2,6-dioxopiperidin-3-yl)-5-iminoisoindolin.

Other specific immunomodula the respective compounds belong to the class of substituted 2-(2,6-dioxopiperidin-3-yl)phthalimides and substituted 2-(2,6-dioxopiperidin-3-yl)-1-occaisonal, such as described in U.S. patent No. 6281230; 6316471; 6335349; and 6476052 and international application WO 98/03502, each of which is incorporated into this description by reference. Specific examples of the compounds have the formula

where

one of X and Y represents C=O, and the other of X and Y represents C=O or CH2;

(i) each of R1, R2, R3and R4independently denotes a halogen atom, alkyl containing from 1 to 4 carbon atoms, or alkoxy containing from 1 to 4 carbon atoms, or (ii) one of R1, R2, R3and R4means-other5and the rest of R1, R2, R3and R4represent a hydrogen atom;

R5denotes a hydrogen atom or alkyl containing 1 to 8 carbon atoms;

R6denotes a hydrogen atom or alkyl containing 1 to 8 carbon atoms, benzyl or halogen atom; provided that R6is different from hydrogen if X and Y denote C=O and (i) each of R1, R2, R3and R4denotes a fluorine atom or (ii) one of R1, R2, R3or R4represents amino.

Some members of this class have the formula

where R1denotes a hydrogen atom or methyl. A separate variant of implementation of the present invention encompasses the use of enantio is Erno pure forms (in particular, optically pure (R)- or (S)-enantiomer) of such compounds.

Other specific immunomodulatory compounds belong to the class of isoindoline disclosed in published applications US patent No. 2003/0096841 and No. 2003/0045552, as well as in WO 02/059106, each of which is incorporated into this description by reference. Individual members have the formula II

and their pharmaceutically acceptable salts, hydrates, solvate, clathrates, enantiomers, diastereomers, racemates and mixtures of stereoisomers, where:

one of X and Y represents C=O and the other represents CH2or C=O;

R1denotes H, (C1-C8)alkyl, (C3-C7)cycloalkyl, (C2-C8)alkenyl, (C2-C8)quinil, benzyl, aryl, (C0-C4)alkyl-(C1-C6)heteroseksualci, (C0-C4)alkyl-(C2-C5)heteroaryl, C(O)R3C(S)R3C(O)OR4, (C1-C8)alkyl-N(R6)2, (C1-C8)alkyl-OR5, (C1-C8)alkyl-C(O)OR5C(O)other3C(S)other3C(O)NR3R3'C(S)NR3R3'or (C1-C8)alkyl-O(CO)R5;

R2denotes H, F, benzyl, (C1-C8)alkyl, (C2-C8)alkenyl or (C2-C8)quinil;

R3and R3'independently denote a (C1-C8)alkyl, (C3-C7)the CEC shall alkyl, (C2-C8)alkenyl, (C2-C8)quinil, benzyl, aryl, (C0-C4)alkyl-(C1-C6)heteroseksualci, (C0-C4)alkyl-(C2-C5)heteroaryl, (C0-C8)alkyl-N(R6)2, (C1-C8)alkyl-OR5, (C1-C8)alkyl-C(O)OR5, (C,-C8)alkyl-O(CO)R5or C(O)OR5;

R4means (C1-C8)alkyl, (C2-C8)alkenyl, (C2-C8)quinil, (C1-C4)alkyl-OR5, benzyl, aryl, (C0-C4)alkyl-(C1-C6)heteroseksualci or (C0-C4)alkyl-(C2-C5)heteroaryl;

R5means (C1-C8)alkyl, (C2-C8)alkenyl, (C2-C8)quinil, benzyl, aryl, or (C2-C5)heteroaryl;

in each case, R6independently denotes H, (C1-C8)alkyl, (C2-C8)alkenyl, (C2-C8)quinil, benzyl, aryl, (C2-C5)heteroaryl or (C0-C8)alkyl-C(O)O-R5or group, R6can be combined with education heteroalkyl group;

n denotes 0 or 1; and

* denotes the carbon atom at the chiral center.

In particular the compounds of formula II, when n is 0, R1means (C3-C7)cycloalkyl, (C2-C8)alkenyl, (C2-C8)quinil, benzyl, aryl, (C0 -C4)alkyl-(C1-C6)heteroseksualci, (C0-C4)alkyl-(C2-C5)heteroaryl, C(O)R3C(O)OR4, (C1-C8)alkyl-N(R6)2, (C1-C8)alkyl-OR5, (C1-C8)alkyl-C(O)OR5C(S)other3or (C1-C8)alkyl-O(CO)R5;

R2represents H or (C1-C8)alkyl; and

R3means (C1-C8)alkyl, (C3-C7)cyclooctyl, (C2-C8)alkenyl, (C2-C8)quinil, benzyl, aryl, (C0-C4)alkyl-(C1-C6)heteroseksualci, (C0-C4)alkyl-(C2-C5)heteroaryl, (C5-C8)alkyl-N(R6)2; (C0-C8)alkyl-NH-C(O)O-R5; And (C1-C8)alkyl-OR5, (C1-C8)alkyl-C(O)OR5, (C1-C8)alkyl-O(CO)R5or C(O)OR5; and the other variables have the same values.

In other specific compounds of formula II, R2represents H or (C1-C4)alkyl.

In other specific compounds of formula II, R1means (C1-C8)alkyl or benzyl.

In other specific compounds of formula II, R1denotes H, (C1-C8)alkyl, benzyl, CH2OCH3CH2CH2OCH3or group

In other variant embodiments of compounds of formula II, R1 refers to a group

where Q denotes O or S, and in each case, R7independently denotes H, (C1-C8)alkyl, (C3-C7)cycloalkyl, (C2-C8)alkenyl, (C2-C8)quinil, benzyl, aryl, halogen atom, (C0-C4)alkyl-(C1-C6)heteroseksualci, (C0-C4)alkyl-(C2-C5)heteroaryl, (C0-C8)alkyl-N(R6)2, (C1-C8)alkyl-OR5, (C1-C8)alkyl-C(O)OR5, (C1-C8)alkyl-O(CO)R5or C(O)OR5or adjacent groups R7can be combined with the formation of substituted bicyclic alkyl or aryl cycle.

In other specific compounds of formula II, R1denotes C(O)R3.

In other specific compounds of formula II, R3means (C0-C4)alkyl-(C2-C5)heteroaryl, (C1-C8)alkyl, aryl or (C0-C4)alkyl-OR5.

In other specific compounds of formula II heteroaryl is pyridyl, furyl or thienyl.

In other specific compounds of formula II, R1denotes C(O)OR4.

In other specific compounds of formula II, the hydrogen atom in the group C(O)NHC(O) may be substituted (C1-C4)alkyl, aryl or benzyl.

Further examples of compounds included in anyclass compounds, include, but not limited to: [2-(2,6-dioxopiperidin-3-yl)-1,3-dioxo-2,3-dihydro-1H-isoindole-4-ylmethyl]amide; (2-(2,6-dioxopiperidin-3-yl)-1,3-dioxo-2,3-dihydro-1H-isoindole-4-ylmethyl)carbamino acid tert-butyl ester; 4-(aminomethyl)-2-(2,6-dioxo-(3-piperidyl))isoindoline-1,3-dione; N-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxo-2,3-dihydro-1H-isoindole-4-ylmethyl)ndimethylacetamide; N-{(2-(2,6-dioxo-(3-piperidyl)-1,3-dixoide-indolin-4-yl)methyl}cyclopropanecarboxamide; 2-chloro-N-{(2-(2,6-dioxo-(3-piperidyl))-1,3-doxasozin-4-yl)methyl}ndimethylacetamide; N-(2-(2,6-dioxo-(3-piperidyl))-1,3-doxasozin-4-yl)-3-pyridinecarboxamide; 3-{1-oxo-4-(benzylamino)isoindoline-2-yl}piperidine-2,6-dione; 2-(2,6-dioxo-(3-piperidyl))-4-(benzylamino)isoindoline-1,3-dione; N-{(2-(2,6-dioxo-(3-piperidyl))-1,3-doxasozin-4-yl)methyl}propanamide; N-{(2-(2,6-dioxo-(3-piperidyl))-1,3-doxasozin-4-yl)methyl}-3-pyridinecarboxamide; N-{(2-(2,6-dioxo-(3-piperidyl))-1,3-doxasozin-4-yl)methyl}heptanone; N-{(2-(2,6-dioxo-(3-piperidyl))-1,3-doxasozin-4-yl)methyl}-2-fullcircle; {Ν-(2-(2,6-dioxo-(3-piperidyl))-1,3-doxasozin-4-yl)carbarnoyl}acetate; N-(2-(2,6-dioxo-(3-piperidyl))-1,3-doxasozin-4-yl)pentanone; N-(2-(2,6-dioxo-(3-piperidyl))-1,3-doxasozin-4-yl)-2-thienylboronic; N-{[2-(2,6-dioxo-(3-piperidyl))-1,3-doxasozin-4-yl)methyl}(butylamino)carboxamide; N-{[2-(2,6-dioxo-(3-piperidyl))-1,3-diocesain the Olin-4-yl)methyl}(octylamine)carboxamide; and N-{[2-(2,6-dioxo-(3-piperidyl))-1,3-doxasozin-4-yl)methyl}(benzylamino)carboxamide.

Finally, other specific immunomodulatory compounds belong to the class of isoindoline disclosed in published patent application U.S. No. 2002/0045643, international application WO 98/54170 and U.S. patent No. 6395754, each of which is incorporated into this description by reference. Individual representatives are the compounds of formula III

and their pharmaceutically acceptable salts, hydrates, solvate, clathrates, enantiomers, diastereomers, racemates and mixtures of stereoisomers, where:

one of X and Y represents C=O and the other represents CH2or C=O;

R represents H or CH2OCOR';

(i) each of R1, R2, R3or R4independently denotes a halogen atom, alkyl containing from 1 to 4 carbon atoms, or alkoxy containing from 1 to 4 carbon atoms, or (ii) one of R1, R2, R3or R4represents nitro or the group-other5and the rest of R1, R2, R3or R4represent a hydrogen atom;

R5denotes a hydrogen atom or alkyl containing 1 to 8 carbon atoms;

R6denotes a hydrogen atom, alkyl containing from 1 to 8 carbon atoms, benzo, a chlorine atom or a fluorine atom;

R' represents R7-CHR10-N(R8R9 );

R7denotes a m-phenylene or p-phenylene or(CnH2n)-, where n has a value from 0 to 4;

each of R8and R9independently denotes a hydrogen atom or alkyl containing 1 to 8 carbon atoms, or R8and R9together form tetramethylene, pentamethylene, hexamethylene or-CH2CH2X1CH2CH2- where X1denotes-O-, -S - or-NH-;

R10denotes a hydrogen atom, alkyl containing from 1 to 8 carbon atoms, or phenyl; and

* denotes the carbon atom at the chiral center.

Other exemplary compounds are the compounds of formula

where

one of X and Y represents C=O, and the other of X and Y represents C=O or CH2;

(i) each of R1, R2, R3or R4independently denotes a halogen atom, alkyl containing from 1 to 4 carbon atoms, or alkoxy containing from 1 to 4 carbon atoms, or (ii) one of R1, R2, R3or R4denotes the group-other5and the rest of R1, R2, R3or R4represent a hydrogen atom;

R5denotes a hydrogen atom or alkyl containing 1 to 8 carbon atoms;

R6denotes a hydrogen atom, alkyl containing from 1 to 8 carbon atoms, benzo, a chlorine atom or a fluorine atom;

R7denotes the-phenylene or p-phenylene or(C nH2n)-, where n has a value from 0 to 4;

each of R8and R9independently denotes a hydrogen atom or alkyl containing 1 to 8 carbon atoms, or R8and R9together form tetramethylene, pentamethylene, hexamethylene or-CH2CH2X1CH2CH2- where X1denotes-O-, -S - or-NH-; and

R10denotes a hydrogen atom, alkyl containing from 1 to 8 carbon atoms, or phenyl.

Other exemplary compounds are the compounds of formula

where

one of X and Y represents C=O, and the other of X and Y represents C=O or CH2;

(i) each of R1, R2, R3or R4independently denotes a halogen atom, alkyl containing from 1 to 4 carbon atoms, or alkoxy containing from 1 to 4 carbon atoms, or (ii) one of R1, R2, R3or R4represents nitro or substituted by an amino group, and the remainder of R1, R2, R3or R4represent a hydrogen atom; and

R6denotes a hydrogen atom, alkyl containing from 1 to 8 carbon atoms, benzo, a chlorine atom or a fluorine atom.

Other exemplary compounds are the compounds of formula

where

one of X and Y represents C=O, and the other of X and Y represents C=O or CH2;

(i) each of R12, R3or R4independently denotes a halogen atom, alkyl containing from 1 to 4 carbon atoms, or alkoxy containing from 1 to 4 carbon atoms, or (ii) one of R1, R2, R3or R4denotes the group-other5and the rest of R1, R2, R3or R4represent a hydrogen atom;

R5denotes a hydrogen atom, alkyl containing from 1 to 8 carbon atoms, or CO-R7-CH(R10)NR8R9where the value of each of R7, R8, R9and R10already defined in the present description; and

R6represents alkyl containing from 1 to 8 carbon atoms, benzo, a chlorine atom or a fluorine atom.

Specific examples of these compounds is of the formula

where

one of X and Y represents C=O, and the other of X and Y represents C=O or CH2;

R6denotes a hydrogen atom, alkyl containing from 1 to 8 carbon atoms, benzo, a chlorine atom or a fluorine atom.

R7represents m-phenylene, p-phenylene or(CnH2n)-, where n has a value from 0 to 4;

each of R8and R9independently denotes a hydrogen atom or alkyl containing 1 to 8 carbon atoms, or R8and R9together form tetramethylene, pentamethylene, hexamethylene or-CH2CH2X1CH2CH2, where X1denotes-O-, -S - or-NH-; and

R10denotes a hydrogen atom, alkyl containing from 1 to 8 carbon atoms, or phenyl.

The most preferred immunomodulatory compounds are 4-(amino)-2-(2,6-dioxo-(3-piperidyl))isoindoline-1,3-dione and 3-(4-amino-1-oxo-1,3-dihydroindol-2-yl)piperidine-2,6-dione. These compounds may be obtained using standard synthetic methods (see, in particular, U.S. patent No. 5635517, which is included in the present description by reference). These compounds can be obtained from the company Celgene Corporation, Warren, NJ. 4-(Amino)-2-(2,6-dioxo-(3-piperidyl))isoindoline-1,3-dione has the following chemical structure

The compound 3-(4-amino-1-oxo-1,3-dihydroindol-2-yl)piperidine-2,6-dione has the following chemical structure:

In another embodiment, the present invention specific immunomodulatory compounds encompass polymorphic forms of 3-(4-amino-1-oxo-1,3-dihydroindol-2-yl)piperidine-2,6-dione, such as form A, B, C, D, E, F, G and H, disclosed in patent publication U.S. No. 2005/0096351 A1, which is incorporated into this description by reference. For example, form A 3-(4-amino-1-oxo-1,3-dihydroindol-2-yl)piperidine-2,6-dione is resolutionand crystalline substance that mo is but to get from systems of nonaqueous solvents. Pattern forms A contains the significant peaks approximately at angles 2θ equal to 8; 14,5; 16; 17,5; 20,5; 24 and 26, and a maximum melting point, determined by the method of differential scanning calorimetry (DSC), is approximately 270°C. Form A weakly hygroscopic or non-hygroscopic and, apparently, is the most thermodynamically stable anhydrous polymorphs 3-(4-amino-1-oxo-1,3-dihydroindol-2-yl)piperidine-2,6-dione discovered to date.

Form B of 3-(4-amino-1-oxo-1,3-dihydroindol-2-yl)piperidine-2,6-dione is palpitation crystalline substance, which can be obtained from different solvents, including, but not limited to, hexane, toluene and water. Pattern of form B contains the significant peaks at angles 2θ of approximately 16, 18, 22 and 27, and has endothermy on the DSC curve at approximately 146 and 268°C, which identify as dehydration and melting in the study on the hot table microscope. The study of the interconversion show that the form B is transformed into the form E in an aqueous solvent system and is transformed into other forms in anhydrous acetone and other systems.

Form C of 3-(4-amino-1-oxo-1,3-dihydroindol-2-yl)piperidine-2,6-dione is prosolution crystalline substance, the cat is PoE can be obtained from the solvent, such, but not limited to, such as acetone. Pattern of form C contains significant peaks at angles 2θ of approximately 15.5 and 25, and has a maximum melting point, determined by the method of differential scanning calorimetry, at approximately 269°C. Form C is not hygroscopic at a relative humidity of less than about 85%, however may become a form of B at higher values of relative humidity.

Form D-3-(4-amino-1-oxo-1,3-dihydroindol-2-yl)piperidine-2,6-dione is a solvated crystalline polymorph, obtained from a mixture of acetonitrile and water. Pattern of form D contains significant peaks at angles 2θ of approximately 27 and 28, and has a maximum melting point, determined by the method of differential scanning calorimetry, at approximately 270°C. Form D or weakly hygroscopic or nephroscopy, but it usually turns into the form B, when it is subjected to stress at high values of relative humidity.

Form E 3-(4-amino-1-oxo-1,3-dihydroindol-2-yl)piperidine-2,6-dione is a double-hydrated crystalline substance, which can be obtained by suspendirovanie 3-(4-amino-1-oxo-1,3-dihydroindol-2-yl)piperidine-2,6-dione in water and on slow evaporation of 3-(4-amino-1-OK, what about the-1,3-dihydroindol-2-yl)piperidine-2,6-dione in aqueous system with a ratio of approximately 9:1 acetone:water. Pattern of form E contains significant peaks at angles 2θ of approximately 20 and 24.5 and 29, and has a maximum melting point, determined by the method of differential scanning calorimetry, at approximately 269°C. Form E can be transformed to the form C in the solvent system acetone base and in form G in the solvent system on the basis of THF. In aqueous solutions of the form E, apparently, is the most stable form. The study desolvatation form E shows that when heated to approximately 125°C. for approximately five minutes, form E can be transformed to the form F.

Form F 3-(4-amino-1-oxo-1,3-dihydroindol-2-yl)piperidine-2,6-dione is resolutionand crystalline substance, which can be obtained by dehydration of form E. Pattern of the form F contains significant peaks at angles 2θ of approximately 19, 19.5 and 25, and has a maximum melting point, determined by the method of differential scanning calorimetry, at approximately 269°C.

Form G of 3-(4-amino-1-oxo-1,3-dihydroindol-2-yl)piperidine-2,6-dione is resolutionand crystalline substance, which can be obtained by suspendirovanie form B and form E in a solvent such as, but not limited to, tetrahydrofuran (THF). Pattern of form G contains is it significant peaks at angles 2θ, approximately equal to 21, 23 and 24.5, and has a maximum melting point, determined by the method of differential scanning calorimetry, at about 267°C.

Form H 3-(4-amino-1-oxo-1,3-dihydroindol-2-yl)piperidine-2,6-dione is a partially hydrated (approximately 0.25 mol) crystalline substance, which can be obtained by keeping the form E at a relative humidity of 0%. Pattern of the form H contains significant peaks at angles 2θ of approximately 15, 26 and 31, and has a maximum melting point, determined by the method of differential scanning calorimetry, at approximately 269°C.

Other specific immunomodulatory compounds that can be used in the methods proposed in the present invention include, but are not limited to, 1-oxo-2-(2,6-dioxo-3-foreperiod-3-yl)isoindolines and 1,3-dioxo-2-(2,6-dioxo-3-foreperiod-3-yl)isoindoline, such as described in U.S. patent No. 5874448 and 5955476, each of which is incorporated into this description by reference. Individual representatives are the compounds of formula

where

Y denotes an oxygen atom or H2and

each of R1, R2, R3or R4independently denotes a hydrogen atom, halogen atom, alkyl, content is ASCII from 1 to 4 carbon atoms, alkoxy containing from 1 to 4 carbon atoms, or amino.

Other specific immunomodulatory compounds suitable for use in the methods proposed in the present invention include, but are not limited to, 2-(2,6-dioxopiperidin-3-yl)-1-occaisonally described in U.S. patent No. 5798368, which is included in the present description by reference. Individual representatives are the compounds of formula

where each of R1, R2, R3or R4independently denotes a halogen atom, alkyl containing from 1 to 4 carbon atoms, or alkoxy containing from 1 to 4 carbon atoms.

Other specific immunomodulatory compounds that can be used in the methods proposed in the present invention include, but are not limited to, 1-oxo - or 1,3-dioxo-2-(2,6-dioxopiperidin-3-yl)isoindoline disclosed in U.S. patent No. 6403613, which is included in the present description by reference. Individual representatives are the compounds of formula

where

Y denotes an oxygen atom or H2,

the first of R1and R2denotes a halogen atom, alkyl, alkoxy, alkylamino, dialkylamino, cyano or carbarnoyl, and the second from R1and R2regardless of the first denotes the atom bodoro is a, halogen atom, alkyl, alkoxy, alkylamino, dialkylamino, cyano or carbarnoyl, and

R3denotes a hydrogen atom, alkyl or benzyl.

Specific examples of the compounds represented by the formula

where

the first of R1and R2denotes a halogen atom, alkyl containing from 1 to 4 carbon atoms, alkoxy containing from 1 to 4 carbon atoms, dialkylamino, in which each alkyl contains from 1 to 4 carbon atoms, cyano or carbarnoyl,

the second of the R1and R2regardless of the first denotes a hydrogen atom, halogen atom, alkyl containing from 1 to 4 carbon atoms, alkoxy containing from 1 to 4 carbon atoms, alkylamino, in which the alkyl contains from 1 to 4 carbon atoms, dialkylamino, in which each alkyl contains from 1 to 4 carbon atoms, cyano or carbarnoyl, and

R3denotes a hydrogen atom, alkyl containing from 1 to 4 carbon atoms, or benzyl. Specific examples include, but not limited to, 1-oxo-2-(2,6-dioxopiperidin-3-yl)-4-methylisoquinoline.

Other compounds that can be used in the methods proposed in the present invention, have the formula

where

the first of R1and R2denotes a halogen atom, alkyl containing from 1 to 4 carbon atoms, alkoxy containing from 1 to 4 atoms is of Pereda, dialkylamino, in which each alkyl contains from 1 to 4 carbon atoms, cyano or carbarnoyl,

the second of the R1and R2regardless of the first denotes a hydrogen atom, halogen atom, alkyl containing from 1 to 4 carbon atoms, alkoxy containing from 1 to 4 carbon atoms, alkylamino, in which the alkyl contains from 1 to 4 carbon atoms, dialkylamino, in which each alkyl contains from 1 to 4 carbon atoms, cyano or carbarnoyl, and

R3denotes a hydrogen atom, alkyl containing from 1 to 4 carbon atoms, or benzyl.

Other specific examples of immunomodulatory compounds that can be used in the methods proposed in the present invention include, but are not limited to, 1-oxo - and 1,3-dioxoindoline, substituted at the 4 - or 5-position indolinone cycle, which is described in U.S. patent No. 6380239 or published patent application U.S. No. 2006/0084815, which is incorporated into this description by reference. Individual representatives are the compounds of formula

where the carbon atom marked With* indicates a chiral center (when n is different from zero, and a value of R1different from the values of R2); one of X1and X2denotes amino, nitro, alkyl containing from one to six carbon atoms, or NH-Z, and the other of X1and X2denotes a hydrogen atom; each of R1and R2independently of the other represents hydroxy or NH-Z; R3denotes a hydrogen atom, alkyl containing from one to six carbon atoms, halogen atom or halogenated; Z represents a hydrogen atom, aryl, alkyl containing from one to six carbon atoms, formyl or acyl containing from one to six carbon atoms; and n is 0, 1 or 2; provided that if X1denotes amino, and n is 1 or 2, R1and R2both represent hydroxy; and their salts.

Other compounds that can be used in the methods proposed in the present invention, have the formula

where the carbon atom marked With* indicates a chiral center when n is different from zero, and a value of R1different from the values of R2; one of X1and X2denotes amino, nitro, alkyl containing from one to six carbon atoms, or NH-Z, and the other of X1and X2denotes a hydrogen atom; each of R1and R2independently of the other represents hydroxy or NH-Z; R3denotes alkyl containing from one to six carbon atoms, a halogen atom or a hydrogen atom; Z represents a hydrogen atom, aryl, or alkyl or acyl containing from one to six carbon atoms; n is 0, 1 or 2.

Specific examples of the compounds, to the which can be used in ways proposed in the present invention include, but are not limited to, 2-(4-amino-1-oxo-1,3-dihydroindol-2-yl)-4-carbamoylmethyl acid and 4-(4-amino-1-oxo-1,3-dihydroindol-2-yl)-4-carbamoylmethyl acid, which have the following structures, respectively, and their pharmaceutically acceptable salt, solvate, prodrugs and stereoisomers

Other exemplary compounds have the formula

where the carbon atom marked With* indicates a chiral center when n is different from zero, and a value of R1different from the values of R2; one of X1and X2denotes amino, nitro, alkyl containing from one to six carbon atoms, or NH-Z, and the other of X1and X2denotes a hydrogen atom; each of R1and R2independently of the other represents hydroxy or NH-Z; R3denotes alkyl containing from one to six carbon atoms, a halogen atom or a hydrogen atom; Z represents a hydrogen atom, aryl, or alkyl or acyl containing from one to six carbon atoms; and n is 0, 1 or 2; and their salts.

Specific examples include, but not limited to, 4-carbarnoyl-4-{4-[(furan-2-ylmethyl)amino]-1,3-dioxo-1,3-dihydroindol-2-yl}butyric acid, 4-carbarnoyl-2-{4-[(furan-2-ylmethyl)amino]-1,3-dioxo-1,3-dihydroindol-2-the l}butyric acid, 2-{4-[(furan-2-ylmethyl)amino]-1,3-dioxo-1,3-dihydroindol-2-yl}-4-phenylcarbamoyloxy acid and 2-{4-[(furan-2-ylmethyl)amino]-1,3-dioxo-1,3-dihydroindol-2-yl}pentanedionato acid, which have the following structures, respectively, and their pharmaceutically acceptable salt, solvate, prodrugs and stereoisomers

Other specific examples of compounds have the formula

where

one of X1and X2represents nitro or NH-Z, and the other of X1and X2denotes a hydrogen atom;

each of R1and R2independently of the other represents hydroxy or NH-Z;

R3denotes alkyl containing from one to six carbon atoms, halogen atom or hydrogen atom;

Z denotes a hydrogen atom, phenyl, acyl containing from one to six carbon atoms, or alkyl containing from one to six carbon atoms; and

n is 0, 1 or 2;

provided that if one of X1and X2denotes nitro, and n is 1 or 2, R1and R2both represent hydroxy; and

if COR2and -(CH2)nCOR1different, the carbon atom marked With* represents a chiral center. Other exemplary compounds have the formula:

where

one of X1and X2about the means alkyl, containing from one to six carbon atoms;

each of R1and R2independently of the other represents hydroxy or NH-Z;

R3denotes alkyl containing from one to six carbon atoms, halogen atom or hydrogen atom;

Z denotes a hydrogen atom, phenyl, acyl containing from one to six carbon atoms, or alkyl containing from one to six carbon atoms; and

n is 0, 1 or 2; and

if COR2and -(CH2)nCOR1different, the carbon atom marked With* represents a chiral center.

Finally, other specific immunomodulatory compounds include, but are not limited to, isoindoline-1-he and isoindoline-1,3-dione substituted in the 2-position of 2,6-dioxo-3-hydroxypiperidine-5-yl, which are described in U.S. patent No. 6458810 included in the present description by reference. Exemplary compounds have the formula

where

the carbon atom marked With* represents a chiral center;

X denotes-C(O)- or-CH2-;

R1represents alkyl containing from 1 to 8 carbon atoms, or-other3;

R2denotes a hydrogen atom, alkyl containing from 1 to 8 carbon atoms, or a halogen atom; and

R3denotes a hydrogen atom,

alkyl containing from 1 to 8 carbon atoms, which is substituted or illegal is Eden with alkoxy, containing from 1 to 8 carbon atoms, halogen atom, amino or alkylamino containing from 1 to 4 carbon atoms,

cycloalkyl containing from 3 to 8 carbon atoms,

phenyl, substituted or unsubstituted alkyl containing 1 to 8 carbon atoms, alkoxy containing from 1 to 8 carbon atoms, halogen atom, amino or alkylamino containing from 1 to 4 carbon atoms,

benzyl, substituted or unsubstituted alkyl containing 1 to 8 carbon atoms, alkoxy containing from 1 to 8 carbon atoms, halogen atom, amino or alkylamino containing from 1 to 4 carbon atoms, or the group COR4in which

R4denotes a hydrogen atom,

alkyl containing from 1 to 8 carbon atoms, which is substituted or unsubstituted with alkoxy containing from 1 to 8 carbon atoms, halogen atom, amino or alkylamino containing from 1 to 4 carbon atoms,

cycloalkyl containing from 3 to 8 carbon atoms,

phenyl, substituted or unsubstituted alkyl containing 1 to 8 carbon atoms, alkoxy containing from 1 to 8 carbon atoms, halogen atom, amino or alkylamino containing from 1 to 4 carbon atoms, or

benzyl, substituted or unsubstituted alkyl containing 1 to 8 carbon atoms, alkoxy containing from 1 to 8 carbon atoms, a halogen atom, aminoil alkylamino, containing from 1 to 4 carbon atoms.

Here the connection or you can buy on a commercial basis, or to receive in accordance with the methods described in the patents or patent publications cited in the present description. In addition, optically pure compounds can be obtained by asymmetric synthesis or obtained by splitting with known disintegrating agents or chiral columns, and other standard methods of synthetic organic chemistry.

Various immunomodulatory compounds contain one or more chiral centers and can exist as racemic mixtures of enantiomers or mixtures of diastereomers. The application covers the pure forms of these compounds, and the use of mixtures of such forms. For example, mixtures comprising equal or unequal amounts of the enantiomers of specific immunomodulatory compounds can be used in methods and compositions that are offered in the present invention. These isomers can be obtained by asymmetric synthesis or by splitting using standard methods, such as chiral chiral column or disintegrating agents. See, in particular, Jacques, J., et al.,Enantiomers, Racemates and Resolutions(Wiley-Interscience, New York, 1981); Wilen, S. H., et al.,Tetrahedron33:2725 (1977); Eliel, E. L.,Stereochemistry of Carbon Compound (McGraw-Hill, NY, 1962); and Wilen, S. H.,Tables of Resolving Agents and Optical Resolutionsp.268 (E. L. Eliel, Ed., Univ. of Notre Dame Press, Notre Dame, IN, 1972).

It should be noted that if there is a contradiction between the depicted structure and the name given to this structure, the depicted structure takes precedence. In addition, if the stereochemistry of a structure or part of a structure is not indicated, for example, bold or dashed lines, it should be assumed that the structure or portion of structure covers all possible for her stereoisomers.

5.9. Introduction PINK cells, placental perfusion solution man or combined natural killer cells

These PINK cells, cells of placental perfusion solution person, combined natural killer cells, a cell population containing cells, or combinations thereof, can be administered to the individual, such as an individual having tumor cells, in particular cancer patient, any well-known from the field of medical equipment acceptable way, which is suitable for the implementation of the introduction of living cells. In various embodiments, implementation of the present invention cells proposed in the present invention may be surgically implanted, introduced by injection, infusion, for example by means of a catheter or syringe, or introduced directly or indirectly into the place, to the / establishment, which you want to restore or increase. In one embodiment of the present invention, these cells are introduced to the individual intravenously. In another embodiment of the present invention, these cells are administered to the individual at the place of location of the tumor, for example, a solid tumor. In a specific embodiment of the present invention, when the individual has a tumor in more than one place, the cells are introduced at least in two places or in all locations of the tumors. In other specific embodiments of the invention cells are proposed in the present invention, or compositions containing these cells, injected oral, intranasal, intraarterially, parenteral, ophthalmologist, intramuscularly, subcutaneously, intraperitoneally, intracerebrally, intraventricular, intracerebroventricular, vnutriobolochechnoe, intracisternal, intraspinal or perispinal. In some specific embodiments, the implementation of the present invention, these cells are delivered via intracranial or vnutriploschadochnyh needles and/or catheters, equipped or not equipped with a pumping device.

These PINK cells, cells of placental perfusion solution person, combined natural killer cells, or combinations thereof, or a population of cells containing such cells, can be administered to an individual in the ideal composition, for example in the form of a matrix, hydrogel, cell skeleton, etc. that include a cell.

In one embodiment of the present invention proposed in the present invention, the cells are plated on a natural matrix, in particular the placental biomaterial, such as the substance of the amniotic membrane. This substance amniotic membrane can be, for example, the amniotic membrane is directly separated from the placenta of a mammal; fixed or heat treated amniotic membrane, almost dried (i.e., containing <20% N2On the amniotic membrane, the membrane chorion, almost dried membrane chorion, almost dried amniotic membrane and membrane chorion, etc. Preferred biomaterials placenta that can do it the stem cells from the placenta, Hariri described in published patent application U.S. No. 2004/0048796, the contents of which are entirely included in the present description by reference.

In another embodiment of the present invention these PINK cells, cells of placental perfusion solution person, combined natural killer cells, or combinations thereof, or a cell population containing cells suspended in a solution of a hydrogel suitable for injection. Suitable hydrogels for like what's compositions include self-assembling peptides such as RAD16. In one embodiment of the present invention the solution of the hydrogel containing cells, allow to harden, for example, in the form, this forms a matrix in which the dispersed intended for implantation of cells. Cells in such a matrix can also be cultivated in such a way that before implantation, cells proliferate mitotically. The hydrogel may be, for example, an organic polymer (natural or synthetic), which cross-crosslinked through covalent, ionic or hydrogen bonds with the formation of three-dimensional structure with an open lattice that holds water molecules, forming a gel. Forming a gel substance include polysaccharides such as alginate or its salts, peptides, polyphosphazene and polyacrylates, which are crosslinked ionic bonds, or block copolymers such as block copolymers of polyethylene oxide and propylene, which are sewn under the action of temperature or pH, respectively. In some embodiments, implementation of the present invention, the hydrogel or the matrix of the present invention is biodegradable.

In some embodiments, implementation of the present invention receive is ain situpolymerization (see, in particular, the published patent application U.S. No. 2002/0022676; Anseth et al.,J. Control Release, 78(1-3):199-20 (2002); Wang et al.,Biomaterials, 24(22):3969-80 (2003)).

In some embodiments, implementation of the present invention the polymers are at least partially soluble in aqueous solutions such as water, buffered salt solutions, or aqueous-alcoholic solutions, and contain carrying a supply side groups or monovalent ion salt. Examples of polymers having acidic side groups that can interact with cations, are poly(phosphazene), poly(acrylic acid), poly(methacrylic acid), copolymers of acrylic acid and methacrylic acid, poly(vinyl acetate) and from sulphonated polymers, such as from sulphonated polystyrene. Can also be used copolymers having acid side groups, which are formed by the interaction of acrylic or methacrylic acid and monomers or polymers of vinyl esters. Examples of the acid groups are groups of carboxylic acids, sulfonic acid group, halogenated (preferably fluorinated) alcohol group, IT group of phenols and acidic Oh groups.

Stem cells from the placenta of the present invention or the corporate culture can be applied to the three-dimensional structure or frame connection and implantin vivo. Such a structure can be implanted in combination with any one or more growth factors, CL is DAMI, medicines and other components, which stimulate the formation of tissues, or in some other way enhance or improve the implementation of the present invention.

Examples of caged compounds that can be used in the present invention include non-woven frame structure, porous foams or self-assembling peptides. Non-woven frame structure can be obtained by using fibers that contain synthetic absorbent copolymer of glycolic acid and lactic acid (for example, PGA/PLA) (VICRYL, Ethicon, Inc., Somerville, N. J.). As the frame structure can also be used foams prepared, for example, of a copolymer of poly(ε-caprolactone)/poly(glycolic acid) (PCL/PGA), which is produced using techniques such as freeze drying or lyophilization (see, in particular, U.S. patent No. 6355699).

Stem cells from the placenta of the present invention can also be applied to, or enter into interaction with physiologically acceptable ceramic substances, including, but not limited to, mono-, di-, tri-, alpha-tri-, beta -, three - and tetracalcium, hydroxyapatite, fluorapatite, calcium sulphate, calcium fluoride, oxides of calcium, calcium carbonates, minimalisti, biologically active glasses such as BIOGLASS®, and mixtures thereof. Porous biocompatible who eroticheskie substances, currently commercially available include SURGIBONE® (CanMedica Corp., Canada), ENDOBON® (Merck Biomaterial France, France), CEROS® (Mathys AG, Bettlach, Switzerland) and mineralized collagen used for implantation of the bone, such as HEALOS™ (DePuy, Inc., Raynham, MA) and VITOSS®, RHAKOSS™ and CORTOSS® (Orthovita, Malvern, Pa.). Three-dimensional structure may be a mixture, the composition or composite of natural and/or synthetic substances.

In accordance with another embodiment of the present invention, the stem cells from the placenta can be applied to felt or enter into interaction with felt, which can be, for example, formed of stranded floss, which is prepared from bioglasses substances, such as copolymers or a mixture of PGA, PLA, PCL, or hyaluronic acid.

In accordance with another embodiment of the present invention, the stem cells from the placenta of the present invention can be applied to frame foams, which can be a composite structure. Such type of foam you can use the mold to impart the desired shape, for example, as part of a specific structure in the body that you want to restore, replace, or increase. In some embodiments, implementation of the present invention a frame structure prior to inoculation of cells according to the present invented the th process, for example, a 0.1 M solution of acetic acid, followed by incubation in polylysine, PBS and/or collagen to strengthen the attachment. The outer surface of the matrix can be modified in order to improve the adherence or enhance cell growth and tissue differentiation, for example, by plasma deposition on the matrix or adding one or more protein (in particular, collagens, elastic fibers, reticular fibers), glycoproteins, glycosaminoglycans (in particular, heparin sulfate, chondroitin-4-sulfate, chondroitin-6-sulfate, sulfate of dermatan, keratin sulfate, and so on), the cell matrix and/or other substances, such as, but not limited to, gelatin, alginates, agar, agarose, resinous substances of vegetable origin, etc.,

In some embodiments, implementation of the present invention frame connection is not thrombogenic or treated with substances that give it nitropropene properties. Such processing and substances can also promote growth or to support the growth of endothelium, migration, and deposition of extracellular matrix. Examples of such substances and treatments include, but not limited to, natural substances such as proteins of the basal membrane, such as laminin and type IV collagen, synthetic substances, such as EPTFE, segmentierung poliuretanovuyu organosilicon compounds such as PURSPAN™ (The Polymer Technology Group, Inc., Berkeley, California). Frame connection can also be an antithrombotic agents such as heparin; frame connections before seeding the stem cells of the placenta can also be processed to change the charge on the surface (in particular, to apply the coating in the plasma).

6. EXAMPLES

6.1. Example 1. The study of the properties of the auxiliary natural killer cells of the placenta, isolated from placental perfusion solution and umbilical cord blood

This example illustrates the isolation and cultivation of natural killer cells from placental perfusion solution man.

The allocation of natural killer cells of the placenta. Natural killer cells isolated from 8 units of placental perfusion solution people (HPP) and 4 units of umbilical cord blood (UCB) using CD56-linked beads. Selection PINK cells is carried out by selection using magnetic beads (Miltenyi Biotec). The placenta after giving birth free from the blood and perfusion with a solution for perfusion (0.9% NaCl injection, which corresponds to the US Pharmacopoeia (catalog number 68200-804, VWR) in an amount of from about 200 to about 750 ml. of the perfusion solution, not held any processing, is collected and removed from the erythrocytes. Mononuclear cells from HPP or the UCB promyvion time buffer solution (RPMI 1640 without phenol red plus 5% FBS) for fluorescently-activated cell sorting (FACS), and then centrifuged with a speed of 1500 rpm for 6 minutes Count the number of cells and clot cells again suspended in 80 μl of buffer 107all particles with 20 μl of CD3 beads (catalog number 130-050-101, Miltenyi). The system is well mixed and incubated for 15 min at a temperature of 4-8°C. Add 1-2 ml of buffer solution of 107all particles, and the mixture is then centrifuged with an acceleration of 300×g for 10 minutes, the Liquid above the sediment completely removed with a pipette. Clot cells again suspended in an amount up to 108cells in 500 ál of buffer and prepare to conduct magnetic separation. Column LS (Miltenyi Biotec) placed in a magnetic field of cell separator MIDIMACS™ (Miltenyi Biotec), washed the column with 3 ml of buffer solution and placed in a column, the suspension of cells and beads. Collect does not contain a label cells CD3‾ who passed through the column and which include natural killer cells, together with 2×3 ml used for flushing the buffer. Cells CD3‾ count, washed once, and then paint with the help of beads CD56 MicroBeads (catalog number 130-050-401, Miltenyi) and distinguish/separate on the same methodology used for the above offices with beads CD3. So gather the population of CD56+CD3‾, which is ready for further and is aleesa. The range of the percentage of natural killer cells is from 3,52 to 11.6 (median: 6,04, average: 5,22) for HPP and from 1.06 to 8.44 for UCB (median: 3,42, average: 4,2). The allocation of natural killer cells from HPP using the beads CD56 gives the population, which has a purity of approximately 80%. Cm. Fig.1. Among the entire population of natural killer cells CD56+, CD3‾, the range of the percentage of natural killer cells CD56+, CD16‾ (i.e., PINK cells) is 56,6 to 87.2 (median: 74,2, average: 65,5) for HPP and 53.7 up to 96.6 (median: 72,8) for UCB. The range of the percentage of natural killer cells CD56+, CD16+is to 43.3 12.8 (median: 25,8, average: 34,5) for HPP and from 3.4 to 46.3 (median: 27,3, average: 33,4) for UCB.

In other experiments, natural killer cells secrete using a set of reagents for conducting negative selection using the magnet, which targets the antigens located on the surface of human blood cells (CD3, CD4, CD14, CD19, CD20, CD36, CD66b, CD123, HLA-DR, glycophorin A). Stored at cryogenic temperature units HPP and UCB thawed and diluted in the ratio 1:1 environment for defrosting (RPMI Media 1640 (catalog number 22400, Gibco) plus 20% serum fetal cow, deactivated by heat treatment (catalog number SH30070.03, Hyclone) and centrifuger the t with a speed of 1500 rpm for 8 minutes The liquid above the precipitate is separated and treated with ammonium chloride in order to further release from red blood cells; each unit is again suspended in approximately 30 ml ice buffer for FACS (RPMI 1640, without phenol red, plus 5% FBS), and then add 60 ml ice ammonium chloride (catalog number 07850, Stem Cell), the solution is vigorously stirred and then incubated on ice for 5 minutes Then mononuclear cells 3 times washed with FACS buffer and centrifuged with a speed of 1500 rpm for 8 minutes Positivt the number of cells and clot cells again suspended in an amount of 5×107living cells per milliliter of the buffer RoboSep Buffer (catalog number 20104, Stem Cell), and added to the cell suspension 0.1 mg/ml solution Gnkazy I (catalog number 07900, Stem Cell), gently mixed with a pipette and before allocation incubated at room temperature for 15 minutes Prior to extraction from the cell suspension by filtering on nylon percolator with a cell size of 40 μm (catalog number 352340, BD Falcon) to remove clumps of cells. The selection is carried out in automatic mode, the device RoboSep (catalog number 20000, Stem Cell) under the program "Human NK Negative Selection 19055 and high recovery" (add 50 ál/ml of the cocktail, add 100 ál/ml of microparticles incubated for 10 and 5 min, separated within 1×2,5 is in) using a set of reagents for enrichment of natural killer cells human Human NK Cell Enrichment Kit (catalog number 19055, Stem Cell), including cocktail EasySep negative selection Negative Selection Human NK Cell Enrichment Cocktail and EasySep magnetic particles Magnetic Microparticles. In this way receive the population of CD56+CD3‾, which is ready for further analysis.

Reproduction of natural killer cells. In General, natural killer cells (NK) multiply the following way. Prepare the initial environment for the culture of natural killer cells, using a modified technique described in Yssel et al.,J. Immunol. Methods72(1):219-227 (1984) and Litwin et al.,J.Exp. Med. 178(4):1321-1326 (1993). In short, the original environment includes IMDM (Invitrogen) with 10% FCS (Hyclone) and contains the following final concentrations of reagents: 35 µg/ml transferrin (Sigma-Aldrich), 5 μg/ml insulin (Sigma-Aldrich), 2×10-5M ethanolamine (Sigma-Aldrich), 1 μg/ml oleic acid (Sigma-Aldrich), 1 mg/ml linoleic acid (Sigma-Aldrich), 0.2 ág/ml palmitic acid (Sigma-Aldrich), 2.5 µg/ml BSA (Sigma-Aldrich) and 0.1 μg/ml of phytohemagglutinin (PHA-P, Sigma-Aldrich). Natural killer cells CD56+CD3‾ again suspended to a concentration of 2.5×105living cells per milliliter source environment plus 200 units/ml IL-2 (R&D Systems) in cell culture, placed in a 24-well plate or in a T-flask. In the original environment as a feeder together add treated with mitomycin C allogeneic PBMC cells and C (cell line chronic myelogenous Le the goat) to a final concentration of 1×10 6per milliliter. NK cells are cultivated in a nutrient medium for 5-6 days at 37°C in atmosphere containing 5% CO2. After 5-6 days, and then every 3-4 days in culture add an equal volume of maintenance medium (IMDM with 10% FCS, 2% serum AB man, antibiotics, L-glutamine and 400 units of IL-2 per milliliter). NK cells are harvested on day 21.

The study of the properties of the auxiliary natural killer cells isolated from the placenta.Having a common donor samples HPP and CB thawed and washed with buffer for FACS (RPMI-1640 with 5% FBS). Then, in accordance with the manufacturer's instructions, natural killer cells enriched with beads CD56, using magnetic separation system ROBOSEP® (StemCell Technologies). Enriched with population of CD56 natural killer cells stained with the following antibodies (if not specifically mentioned, are supplied by BD Bioscience) to determine immunophenotype: against CD56 conjugated with PE-Cy-7, against CD3 APC-Cy7, anti CD16 FITC, against NKG2D APC, against NKp46 APC, against CD94 PE(R&D), against NKB1 PE5and against KIR-NKAT2 PE. CD94, NKG2D and NKp46 not contain markers, or show reduced expression in the precursor NK cells, but are present in fully differentiated NK cells. Cm. Freud et al., "Evidence for Discrete States of Human Natural Killer Cell DifferentiationIn Vivo,"J. Exp.Med.203(4):1033-1043 (2006); Eagle & Trowsdal, "Promiscuity and the Single Receptor: NKG2D,"Nature Reviews Immunologypublished on web site online 3 August 2007; Walzer et al., "Natural Killer Cells: From CD3‾NKp46+to Post-Genomics Meta-Analyses,"Curr.Opinion Immunol.19:365-372 (2007). As indicated in table 1, expression of KIR3DL1, KIR2DL2/L3, NKG2D, NKp46 and CD94 differ slightly between the enriched population of cells CD56+from HPP and HLA-compatible population of cells CD56+from umbilical cord blood (CB).

6.2. Example 2: a Study of auxiliary natural killer cells of the placenta separated from the United placental perfusion solution and umbilical cord blood

Mononuclear cells from umbilical cord blood and placental perfusion solution (combo) from a single donor are mixed and washed once with FACS buffer (RPMI-1640 with 5% FBS) and spend the definition immunophenotype using the antibodies listed in table 2 on BD FACSCanto (BD Biosciences). The resulting data analyzed using the software FlowJo software (Tree Star).

Table 2
The list of antibodies, which are used to study immunophenotype
NameProducerCatalog number
FITC against hu CD3BD Bioscience 555332
FITC against hu CD3Miltenyi130-080-401
APC-Cy7 against hu CD3BD Bioscience557832
FITC against hu CD16BD Bioscience555406
PE-Cy5 against hu CD16BD Bioscience555408
PE against hu CD56BD Bioscience555516
PE against hu CD56Miltenyi130-090-755
PE-CY5 against hu CD56BD Bioscience555517
PE-Cy7 against hu CD56BD Bioscience557747
PE against hu CD94R&DFAB-1058P
PE against hu KIR-NKAT2 (2DL2/L3)BD Bioscience556071
PE against hu NKB1 (3DL1)BD Bioscience555967
APC against hu NKG2D BD Bioscience558071
APC against hu NKp46BD Bioscience558051
PE against hu CD226BD Bioscience559789
PE against hu NKp44BD Bioscience558563
PE against hu NKp30BD Bioscience558407
PE against hu 2B4BD Bioscience550816
the isotype IgG1 FITC mouseBD Bioscience340755
the FITC isotype IgG2b mouseBD Bioscience556577
the PE isotype IgG1 mouseBD Bioscience340761
the PE isotype IgG2b mouseBD Bioscience555743
the isotype IgG1 PerCP mouseBD Bioscience340762
the isotype PE-Cy5 IgG2b mouseBD Bioscience555744
from the Fe APC IgG1 mouse BD Bioscience340754
the APC isotype IgG2a mouseBD Bioscience555576
the isotype APC-Cy7 IgG1 mouseBD Bioscience348802
the isotype PE-Cy5 IgG1 mouseBD Bioscience348798

The study immunophenotype NK cells placental NK cells and peripheral blood (PB).Natural killer cells can be divided into two main groups: NK cells CD56+CD16+and cells CD56+CD16‾. NK cells CD56+CD16+have a lot of cytolytic granules and show high expression of CD16, and therefore capable of causing mediated by antibodies cell-mediated cytotoxicity (ADCC). In contrast, NK cells CD56+CD16‾ have very little cytolytic granules, show low expression or not Express CD16, however, when activated, are capable of producing cytokines and chemokines. Individual natural killer cells demonstrate a diverse set of combinations of activating and inhibitory receptors, including immunoglobulinlike receptors killer cells (KIRs, in particular KIR3DL1 and KIR2DL2/3), the natural cytotoxicity receptors NCRs (in particular, NKp30, NKp44 and NK46), similar to lectins receptors killer cells (KLRs; in particular CD94, NKG2D), 2B4 and CD226.

FACS analysis is performed for natural killer cells of the placenta and natural killer cells in peripheral blood using fluorescently-linked monoclonal antibodies against specific receptors of natural killer cells. Among the studied 11 subgroups of natural killer cells obtained values for the number of cells in seven of the 11 subgroups of natural killer cells (CD3‾CD56+CD16‾, CD3‾CD56+CD16+, CD3‾CD56+KIR2DL2/3+, CD3‾CD56+NKp46+, CD3‾CD56+NKp30+, CD3‾CD56+2B4+and CD3‾CD56+CD94+) show a significant difference (p<0,05) between natural killer cells of the placenta and natural killer cells in peripheral blood (responsible for 64% of the variation) (table 3A; see also tables 3B and 3C).

Tables 3B and 3C show the study of the phenotype of NK cells CD3‾CD56+CD16‾ NK cells and CD3‾CD56+CD16+16 units combined umbilical cord blood and placenta of man from a single donor (combo) and 13 units of peripheral blood (PB) in a separate experiment.

60.9% of the natural killer cells of the placenta are cells CD56+CD16‾ (secondary nature of the performance communications killer cells of the placenta, PINK cells) while only 21.4 per cent of natural killer cells in the peripheral blood cells are CD56+CD16‾. After cultivation in a nutrient medium within 21 days percentage in four of the 11 subgroups of natural killer cells (CD3‾CD56+KIR2DL2/3+, CD3‾CD56+NKp46+, CD3‾CD56+NKp44+and CD3‾CD56+NKp30+) shows a significant difference (p<0,05) between natural killer cells of the placenta and peripheral blood (table 4).

In addition, in a separate experiment show that after growing in a nutrient medium within 21 days of NK cells placental and peripheral blood exhibit a unique profile of cytokines, in particular IL-8, which is determined using Luminex fluorescent analysis (table 5).

Profiling microRNA natural killer cells of the placenta and natural killer cells in peripheral blood,. Dedicated or reproduced natural killer cells used for preparation of microRNA (miRNA) using a set of reagents for the identification of microRNA MIRVANA™ miRNA Isolation Kit (Ambion, catalog number 1560). Natural killer cells (from 0.5 to 1.5×106cells) destructuring in denaturing buffer for lysis. Then the samples are extracted with a mixture of acid-phenol + chloroform to you is elite RNA, significantly enriched samples of small RNA. Add 100% ethanol so that the concentration of ethanol in the sample was 25%. When the mixture lysate/ethanol flow through a funnel with a glass filter beds, large RNA immobilized, and small RNA is collected in the filtrate. Then the concentration of ethanol in the filtrate increased to 55% and the mixture is passed through a glass filter, on which are immobilized a small RNA. These RNA is washed several times and elute solution with low ionic strength. The concentration and purity of extracted small RNA determined by measuring their absorption at the wavelength of 260 and 280 nm.

miRNAs, which, as shown, is unique to the PINK cells are shown in table 6. It was found that one miRNA, designated as a has-miR-199b, unique to NK cells in peripheral blood.

Definition immunophenotype grown in a nutrient medium NK cells of the placenta and rekultivirovannyh NK cells. A full range of properties grown in a nutrient medium PINK cells determined by conducting extensive research immunophenotype and study of cytotoxicity. To determine the phenotype of propagated NK cells to analyze the expression of receptors of natural killer cells (NKRs), such as KIRs, NKG2D, NKp46, NKp44 and 2B4. The study of the cytotoxicity of avodat, marking the tumor cells (cells C) using RCN, and then for 4 h cultivate together with PINK cells. Starting from day 0 to day 21 the expression of NKG2D increases with 60,9%±4.8% to 86%±17.4% (p value 0,024); the expression of NKp46 increases from 10.5%±5.4% to 82.8% of±9.0% (p value 0,00002); the expression of NKp44 increases from 9.6%±6.5% to 51.6 per cent±27,5% (p value of 0.022); and the expression of 2B4 reduced from 13.0% in±7,1% to 0.65%±0,5% (p 0,009) (table 7). In these culture conditions inhibitory KIRs, including KIR3DL1 (immunoglobulinemia receptor killer cell, three domains, long cytoplasmic tail 1, the inhibitory receptor) and KIR2DL2/L3 (immunoglobulinemia receptor killer cell, two domains, long cytoplasmic tail, 2 and long cytoplasmic tail, 3; inhibitory receptors) remain unchanged during propagation within 21 days. In addition, changes in the expression of NKRs correlated with a marked increase in cytolytic activity on day 21, compared to the 14th day, compared to cells C (63%±15% vs. 45%±4%, p value of 0.0004). The data obtained allowed us to identify putative markers of NK cells, which are well correlated with the cytotoxic activity of natural killer cells.

Determination of membrane proteomic profile grown in a nutrient medium NK cells of the placenta and wires is the R in the medium of NK cells in peripheral blood using methods of immobilization using lipids by LC/MS with a linear ion trap.

Purification of a membrane protein:Natural killer cells of the placenta from the combined placental perfusion solution and umbilical cells, and natural killer cells PB, which is grown in a nutrient medium for 21 days, incubated for 15 min with a solution of a cocktail of protease inhibitors (P8340, Sigma Aldrich, St. Louis, MO; contains 4-(2-amino-ethyl)benzene-sulfonyl fluoride (AEBSF), pepstatin a, E-64, bestain, leupeptin and Aprotinin without chelating metals) before lysis of the cells. The cells are then subjected to lysis by adding 10 mm HCl solution, which does not contain detergents, and centrifuged for 10 min with an acceleration of 400×g in order to obtain a bunch of cells and remove cores. The supernatant after removal of nuclei transferred into a test tube of ultracentrifuge and centrifuged in an ultracentrifuge WX80 with the rotor T-1270 (Thermo Fisher Scientific, Asheville, NC) with acceleration 100000×g for 150 min, you get a bunch of membrane protein.

Receiving, immobilization and fermentation by proteoliposome:Clot membrane protein is washed several times with buffer NANOXIS® (10 mm Tris, 300 mm NaCl, pH 8). Clot membrane protein suspended in 1.5 ml buffer NANOXIS®, and then under ice cooling is subjected to ultrasonic processing for ultrasonic installation with a nozzle VIBRA-CELL™ VC505 (Sonics &Materials, Inc., Newtown, CT) for 20 min Size proteoliposome opredelyayushchaya using dye FM1-43 (Invitrogen, Carlsbad, CA) and get a visual image using a fluorescent microscope. The protein concentration in suspension by proteoliposome determined by BCA analysis (Thermo Scientific). Proteoliposome then Inuktitut in LPI™Flow Cell (Nanoxis AB, Gothenburg, Sweden) using standard pipette with a tip and leave for immobilization for 1 hour. After immobilization conduct a series of operations of washing and directly in LPI™Flow Cell Inuktitut 5 μg/ml of trypsin (Princeton Separations, Adelphi, NJ). The granules incubated over night at 37°C. Then trypticase peptides elute from the granulate and absoluut using cartridge Sep-Pak cartridge (Waters Corporation, Milford, MA).

Fractionation on a column with a strong cationic exchange: Trypticase peptides restore in the solution of 0.1% formic acid/water and placed in a column with a strong cation exchange (SCX) TOP-TIP™ (PoIyLC, Columbia, MD), the tip of the pipette, fill 30 μm nozzle from Polyalphaolefine. Peptides elute from SCX TOP-TIP™ in a stepwise gradient-based buffer ammonium formate, pH of 2.8 (10 mm-500 mm). Each SCX fraction is dried with system speed-vac and re-restore 5% acetonitrile, with 0.1% formic acid in preparing for the subsequent analysis of LC/MS.

LTO LC/MS analysis with a linear ion trapEach SCX fraction was separated on a column of 0.2 mm × 150 mm, 3 μm 200 Å MAGIC C18 (Michrom Bioresources, Inc., Auburn,CA), directly coupled with axial source for electrospray ionization in nanobotic with desolately in vacuum (ADVANCE) (Michrom Bioresources, Inc.), using gradient elution within 180 min (buffer A: water with 0.1% formic acid; Buffer b: acetonitrile, with 0.1% formic acid). Source ADVANCE allows you to achieve a sensitivity comparable to that of traditional ion trap high capacity nanoESI, but to work at a considerably higher flow rate, component 3 ál/min Erwerbende peptides analyzed by mass spectrometer with LTO linear ion trap (Thermo Fisher Scientific, San Jose, CA), which used ten-dependent data MS/MS scans after a full scan of each mass spectrum.

BioinformaticsSix RAW files corresponding to 6 salt fractions that are collected for each cell line tumor cells (AML, CML), is studied by means of a single search against databases IPI Human Database using the SEQUEST algorithm, using an automated workplace SORCERER™ SOLO™ (Sage-N Research, San Jose, CA). Ask 1.2 at. unit mass for the permissible deviations of the mass of the peptide, oxidation of methionine take for differential modification, and carbamidomethylation mistaken for a static modification. For sorting and analysis of proteomic data for membranes used in the comfort variant Scaffold software Trans-Proteomic Pipeline (TPP). Proteins allow for analysis if they identify with a peptide probability of 95%, a protein 95% probability and 1 unique peptide. Comparison of sets of proteomic data for membranes is carried out using the method of Perl scripts designed specifically for the company you represent the inventors.

The analysis shows that the identified 8 membrane proteins grown in a nutrient medium NK cells, which are unique with respect to membrane proteins identified from NK cells in peripheral blood. Cm. table 8. In addition, 8 of membrane proteins identified from NK cells in the peripheral blood, which are unique with respect to grown in a nutrient medium NK cells of the placenta. Cm. table 8. It was found that only 10 of the identified membrane proteins are distributed among cultured NK cells of the placenta, and among NK cells in peripheral blood.

Table 8
Proteins that are specific for NK cells of the placentaProteins that are specific for NK cells in peripheral blood
Aminopeptidase NPredecessor receptor 4 fibroblast growth factor
Apolipoprotein ENucleotide-4-like immunoabsorbant protein 1
Protein 1, interacts with atropine-1The precursor integrin alpha-L
Annexin inx-3The precursor integrin beta-2
The precursor integrin alpha-2The predecessor of integrin beta-4
The precursor integrin beta-5The precursor membrane lytic moreintrospective D
The precursor glycoprotein GP49B the surface of mast cellsLinking existieren protein-related protein 8
Receptor 1 ryanodineThe predecessor of perforin 1

6.3. Example 3: Cytotoxicity of natural killer cells against tumor cells

This example shows that the auxiliary natural killer cells of the placenta have cytotoxicity against tumor cells. PINK cells from HPP cytotoxic against cells of acute myelogenous leukemia, which confirms the analysis of cytotoxicity and Luminex analysis of the cytokine secretion of NK cells.

In the analysis of the cytokine secretion of enriched beads CD56 natural killer cells from HPP mixed with cells KG-1a acute myelogenous leukemia in a 1:1 ratio. After incubation for 24 h, collect the supernatant and analyze Luminex secretion of IFN-γ and GM-CSF. Elevated levels of IFN-γ and GM-CSF were observed after 24-hour incubation CD56-enriched cells HPP cells KG-1a, as shown in Fig.2.

Cytotoxicity PINK cells

In the analysis of cytotoxicity using PINK cell tumor target cells mark Succinimidyl air carboxyfluorescein (CFSE). CFSE is a vital dye that is non-toxic towards cells and is distributed between daughter cells during cell division. The cells are then placed on a 96-well tablets with a U-shaped bottom for tissue cultures and incubated with freshly isolated PINK cells CD56+CD16‾ with ratios of effector-target (E:T) equal to 20:1, 10:1, 5:1 and 1:1 in RPMI medium 1640, supplemented with 10% FBS. After 4 h incubation, the cells are harvested and examined by flow cytometry for the presence of CFSE. The number of target cells, isolated from the culture without NK cells, used as a reference. Cytotoxicity is defined as: (1-CFSEsample/CFSEcontrol)*100%. Significant cytotoxicity against op is halewyn cells observed in the ratio of 20:1. Cm. Fig.3.

The sensitivity of tumor cells to grown in a nutrient medium PINK cells

Analysis by the release of lactate dehydrogenase (LDH).Analysis on release of LDH carried out with the help of a set of reagents for the colorimetric determination of cytotoxicity CYTOTOX 96® (Promega, catalog number G1780). This analysis is grown in a nutrient medium NK cells, which are a combination of cells CD56+CD16‾ cells and CD56+CD16+isolated from compatible with each other HPP/UCB are cells effectors, and tumor cells are target cells. Cell effectors and target cells are placed in 96-well tablets with a U-shaped bottom for tissue cultures and incubated at various ratios of effector-target (E:T) in 100 μl of RPMI medium 1640 without phenol red (Invitrogen, catalog number 11835-030), supplemented with 2% serum AB man (Gemini, catalog number 100-512). Culture incubated for 4 h at 37°C in 5% CO2. After incubation 50 μl of supernatant is transferred onto the tablet for carrying out enzymatic analysis, and the LDH activity is determined in accordance with the manufacturer's recommendations, and the absorption is measured at a wavelength of 490 nm in a plate reader for ELISA (Synergy HT, Biotek). The degree of cytotoxicity calculated in accordance with the following equation: % Cytotoxicity = (value for the image is as - An arbitrary value for effector - an Arbitrary value for the target)/(Maximum value for the target is an Arbitrary value for the target)*100%.

Some tumor cells can more easily interact with NK cells than others. To study the sensitivity of tumor cells to grown in a nutrient medium PINK cells are conducting a study using analysis of LDH release for twelve different lines of cancer cells, which grow together with the PINK cells. 12 lines of tumor cells include chronic myelogenous leukemia person (CML), lymphoma, retinoblastoma (RB) and multiple myeloma (MM) (table 9). The cytotoxicity of NK cells was determined by analysis on release of LDH after a 4-hour joint cultivation in a nutrient medium.

Table 9
Data from ATCC cell lines
NameDescription
CCRF-CEMThe human leukemia
KG-1Acute myeloid leukemia person
KG-1AAcute myeloid leukemia person
K562Chronic myeloid leukemia person
KU812Chronic myeloid leukemia person
U-937Histiocytoma lymphoma person
WERI-RB-1Retinoblastoma man
HCC2218Breast cancer man
RPMI8226Multiple myeloma man
HCT116Colorectal carcinoma person
HT29Colorectal adenocarcinoma man
U266Multiple myeloma man

When the ratio of effector to target (E:T) equal to 10:1, significant cytotoxicity grown in a nutrient medium PINK cells was observed compared to cells K562 (CML) with the value of 88.6%±5.6% for cells U937 (lymphoma) with the value of 89.2%±9.8 per cent, to cells WERI-RB-1 (RB) with the value 73,3%±11.8 percent, to RPMI8226 cells (MM) size 61,3%±1.3% and the cells U266 (MM) size 57,4%±4.7% of (table 10).

Increase cytotoxicity PINK cells by treatment with lenalidomide and pomalidomide

p> Isolation and purification of RNA.From dedicated or propagated NK cells using a set of reagents RNAQUEOUS®-4PCR (Ambion, catalog number AM1914) receive RNA. In short, NK cells (from 0.5 to 1.5×106cells) subjected to lysis in the designated holding lysis solution guanidine. The sample lysate is then mixed with ethanol and applied to the filter of silicon oxide, which selectively and quantitatively binds mRNA and larger RNA ribosomes; very small RNA, such as tRNA and 5S ribosomal RNA not bound quantitatively. The filter is then washed to remove residual DNA, protein and other contaminants, and elute RNA nuclease-free water, which contains traces of add for chelation of heavy metals. The filter made of silicon oxide is set in a small cartridge that will fit in not containing the RNase tube microcentrifuge that are supplied with the reagents. The sample lysate, washing solutions and solutions after elution passed through the filter using a centrifuge or by applying a vacuum pressure. After elution of the filter RNA treated with pure Dnazol 1, which comes with a set of reagents to remove trace amounts of DNA. Finally, Tenkasu and divalent cations are removed by means of a reagent, also supply pax is with the reagents. The concentration and purity of the selected RNA, determined by measuring the absorption at a wavelength of 260 nm and 280 nm.

Analysis by real-time quantitative PCR in real time (qRT-PCR).Selected RNA can then be used to synthesize cDNA using a set of reagents for reverse transcription TAQMAN® (Applied Biosystems, catalog number N8080234) with subsequent analysis by real-time quantitative PCR in real time using the 7900HT Fast Real-Time PCR and immune matrices person (Applied Biosystems, catalog number 4370573) and matrix microRNA person (Applied Biosystems, catalog number 4384792).

Lenalidomide and pomalidomide are chemical analogs of thalidomide with enhanced anti-cancer and anti-inflammatory activities. To determine whether lenalidomide and pomalidomide to enhance the cytotoxicity PINK cells,ex vivocultured PINK cells (day 19) previously treated with lenalidomide or pomalidomide within 24 hours with subsequent joint cultivation in a nutrient medium with target cells cell lines of colorectal carcinoma HCT-116. Treated with lenalidomide NK cells exhibit cytotoxicity was 42.1%, and treated pomalidomide PINK cells exhibit cytotoxicity to 47.4%, while the control NK cells not exposed to processing, show only the cytotoxicity of 24.3%.

the analysis by real-time quantitative PCR in real time (qRT-PCR) and flow cytometry shows what caused pomalidomide increase cytotoxicity of NK cells correlates with increased expression of a gene (an increase of 60%±1.7%) are of granzyme B (GZMB) (table 11) and with a high percentage GZMB-positive NK cells (increase of 25%). In addition, the expression of GM-CSF increases in the treated with lenalidomide (an increase of 232%±1,6%) and pomalidomide (increase 396%±0,3%) PINK cells (table 11A, 11B).

Table 11A, 11B. Analysis of qRT-PCR, treated with lenalidomide and pomalidomide grown in a nutrient medium PINK cells, compared with untreated cells. 11A: Fold change in gene expression between treated with lenalidomide and not treated with lenalidomide models for named genes. To determine whether multiple changes in the treated with lenalidomide and not treated with lenalidomide samples using paired t-test. 11B: Fold change in gene expression between treated pomalidomide and not processed pomalidomide samples for 25 named genes. To determine whether multiple changes in the treated and not treated samples using paired t-test.

BAX - BCL-2-associated protein X
CCL5 - ligand 5 chemokine (motif C-C)
CCR5 is a receptor of the chemokine (motif C-C)
CSF2 colony stimulating factor 2 (granulocyte-macrophage)
FAS - superfamily genes of the TNF receptor, member 6
GUSB - glucosidase is a β
IL2RA - receptor-α interleukin 2
TNFSF18 - superfamily genes of the receptor of tumor necrosis factor, member 18

ACTB - β-actin
BAX - BCL-2-associated protein X
CCL2 - ligand 2 chemokine (motif C-C)
CCL3 - ligand 3 chemokine (motif C-C)
CCL5 - ligand 5 chemokine (motif C-C)
CCR5 receptor 5 chemokine (motif C-C)
CSF1 colony stimulating factor 1 (macrophage)
CSF2 colony stimulating factor 2 (granulocyte-macrophage)
ECE1 - endothelin-converting enzyme 1
FAS - superfamily genes of the TNF receptor, member 6
GNLY - granulysin
GUSB - glucuronidase-β
GZMB - Grasim In (Grasim 2, cytotoxic T-lymphocyte-associated carinatherese 1)
IL1A - α interleukin 1
IL2RA - receptor-α interleukin 2
IL8 - interleukin 8
IL10 - interleukin 10
LTA - lymphotoxin α (TNF superfamily, member 1)
PRF1 - perforin 1 (forming pores protein)
PTGS2 - prostaglandin-endoperoxide-synthase 2 (prostaglandin G/H synthase and cyclooxygenase)
SKI - homolog of the viral oncogene v-ski sa is coma (bird)
NBX21 - T-box 21

Cytotoxicity of combined natural killer cells

In a separate analysis of cytotoxicity grown in a nutrient medium NK cells that are taken from a single donor samples of cord blood and placental perfusion solution, cells are the effectors, and the tumor cells are the target cells. Tumor cells labeled with PKH26 (Sigma-Aldrich, catalog number PKH26-GL) (see, in particular, Lee-MacAry et al.,J.Immunol. Meth. 252(1-2):83-92 (2001)), which is embedded in the cytoplasmic membrane of the cell due to its lipophilic aliphatic residue, and then placed on a 96-well tablets with a U-shaped bottom for tissue cultures and incubated with grown in a nutrient medium NK cells at various ratios of effector-target (E:T) in 200 μl of RPMI medium 1640, supplemented with 10% FBS. The culture is incubated at 37°C in an atmosphere of 5% CO2within 4 hours After incubation, the cells are harvested and culture add TO-PRO-3 (Invitrogen, catalog number T3605), not able to penetrate through the membrane dye DNA to a final concentration of 1 μm, and then spend the FACS analysis using a BD FACSCanto. Cytotoxicity is expressed as the percentage of dead cells (PKH26+TO-PRO-3+among all tumor target cells PKH26+.

In this analysis zitotoksicnostthew K562 chronic myeloid lymphoma (CML) man mark with PKH26, which is embedded in the cytoplasmic membrane of the cell, and placed on a 96-well tablets with a U-shaped bottom for tissue cultures. NK cells of the placenta (combined) or NK cells in the peripheral blood, which are cultivated in a nutrient medium for 21 days, mixed with K562 cells at ratios of effector to target (E:T) equal to 10:1, 5:1, 2,5:1 1.25:1 in RPMI medium 1640, supplemented with 10% vol./about. FBS. After incubation for 4 h, the cells are harvested and cell culture add TO-PRO-3, and then examined by flow cytometry for the presence of PKH26 and TO-PRO-3. Cytotoxicity is expressed as the percentage of dead cells PKH26+TO-PRO-3+among all tumor target cells PKH26+. As NK cells of the placenta, and NK cells in peripheral blood showing significant toxicity towards cells C for all investigated relations E:T (Fig.4). A significantly higher toxicity of NK cells of the placenta than NK cells from peripheral blood, compared to cells C observed at two ratios (E:T of 10:1 and 5:1 (Fig.4).

6.4. Example 4: Cytotoxicity of placental perfusion solution person in relation to tumor cells.

This example shows that the cells of the placental perfusion solution person possess cytotoxicity against tumor cells and the cytotoxicity of the combination nucleated to etock of HPP (TNC-HPP) against KG-1a above, than cytotoxicity TNC, selected from a compatible UCB. The combination of nucleated cells from the HPP or umbilical cord blood (UCB) is mixed with cells KG-1a ratio 1:1, 5:1, 10:1, 20:1 or 100: 1. After incubation for 24 h or 48 h, cells are collected and examined in the presence of CFSE using FACS analysis (BD FACSCanto, BD Bioscience). As a control used separately grown in a nutrient medium tumor cells. Cytotoxicity is defined as: (1 - CFSEsample/CFSEcontrol)*100%. Significant cytotoxicity was shown at a ratio of 100:1. Cm. Fig.5.

In a separate experiment, the cytotoxicity of the population of nucleated cells from HPP compared with the cytotoxicity of the population of nucleated cells from umbilical cord blood. Compatible TNC-HPP or UCB mixed with cells KG-1a ratio 0,78:1, 1,56:1, 3,12:1, 6,25:1, 12,5:1, 25:1, 50:1 or 100:1. TNC-HPP show consistently higher cytotoxicity for all the compared ratios, compared with UCB cytotoxicity. Cm. Fig.6.

In another experiment for 24 h before incubation with cells KG-1a cells (TNC)-HPP stimulated with 100 units/ml or 1000 units/ml IL-2, while HPP grown in RPMI medium, used as a control. When the ratio of NK cells to cells KG-1a, equal to 6.25 or more of IL-2 enhances the cytotoxicity TNC-HPP. Cm. Fig.7.

The experiments continue, adopting a broader spectrum of the tumor cells, as indicated in table 12, using 5×105cells HPP and 1×104tumor cells.

Table 12
Types of tumor cells, was investigated on the cytotoxic action of placental perfusion solution
HCC2218Primary carcinoma of the epithelium of the ducts man
CCRF-CEMThe human leukemia
J. RT3 to T3.5Acute T-cell leukemia human
K562Chronic myeloid leukemia person (CML)
KG-1Acute myeloid leukemia person
KG-1AAcute myeloid leukemia person (AML)
KU812The human leukemia (CML)
NCI-H1417Lung carcinoma person
SNU-CIAdenocarcinoma of the colon of a person
U-937Histiocytoma lymphoma person
WERI-RB-1Retinoblastoma man
HCT116Colorectal carcinoma person
HT29Colorectal adenocarcinoma man
U266Myeloma man

When the HPP cells and tumor cells together is grown in a nutrient medium for 24 h or 48 h with a ratio of 50:1, HPP cells show significant toxicity towards tumor cells. For both of these time periods culturing in a nutrient medium leads to the death of more than 50% of tumor cells. Cm. Fig.8A and 8B.

6.5. Example 5: Production of cytokines by cells of the placental perfusion solution person when exposed to tumor cells

To determine the primary mechanism of action, which is responsible for mediating efficient antileykemichesky effects cells HPP, analyze the profile of the release of cytokines by cells of the HPP, which is grown in a nutrient medium together with cell lines, and compared with the profile of the release of cytokines by UCB cells at different points in time, using repetitive analyses on Luminex method.

The supernatant liquid, which is collected after incubation, the item will gorhaut analysis by Luminex method, to determine the concentration of IFN-γ, TNF-α and GM-CSF (catalog number HCYTO-60K-03, Millipore). These three cytokines are associated with cytoxicity NK cells (see, in particular, Imai et al.,Blood 2005, 106(1):376-83). Conduct quantitative RT-PCR to study the expression of IFN-γ, TNF-α and GM-CSF, using the device Applied Biosystems 7900HT FAST and primers. Growing conditions are the same as for joint cultivation analysis for cytotoxicity as described above. The concentration of cytokines was determined by analysis on the Luminex method.

It was found that the secretion of IFN-γ, TNF-α and GM-CSF from HPP cells grown in a nutrient medium together with tumor cells, significantly higher than the secretion of IFN-γ, TNF-α and GM-CSF cells from UCB. In one experiment the HPP cells mixed with cells KG-1a relations, equal 0,78:1, 1,56:1, 3,12:1, 6,25:1, 12,5:1, 25:1, 50:1 or 100:1, both in the presence and absence of 100 units of IL-2. TNC-HPP show steady increase production of IFN-γ in the presence of IL-2, compared with the absence of IL-2. It was shown that 24 h the levels of IFN-γ increase approximately 5-26 times (median: 16 times); 48 h approximately 3-65 times (median: 27 times), which is consistent with the results obtained in the study of cytotoxicity. Cm. Fig.9.

In another experimentee for 24 h before incubation with cells KG-1a cells (TNC)-HPP stimulated with 100 units/ml or 1000 units/the l IL-2, while HPP grown in RPMI medium, used as a control. Cells HPP or compatible UCB cells incubated for 24 h with or without IL-2 before you grow together with the cells KG-1a. The secretion of IFN-γ is the most enhanced in cells HPP, which grow together with K and KG-1a. When cells HPP treated with 100 units/ml IL-2, the cytotoxicity of cells HPP against KG-1a increased after 24 h and 48 h After treatment with IL-2 level secretion of IFN-γ in cells HPP higher than the level of secretion of IFN-γ in a compatible UCB cells. Higher expression of IFN-γ is confirmed by the analysis of the RT-PCR cells from a compatible HPP and UCB. The results show that cells HPP are more antileykemichesky activity, compared with UCB cells, and indicated greater activity is associated with a significant increase in production of IFN-γ.

The production of IFN-γ in the HPP cells and in cells of umbilical cord blood in joint cultivation in a nutrient medium with the above lines of tumor cells analyzed using lines of tumor cells, shown above in table 1. The HPP cells and tumor cells grow together within 24 h or 48 h with a ratio of 50:1 using a 104tumor cells and 5×105cells HPP. For cell lines CCRF-CEM, J. RT3 to T3.5, K562, KG1, KG-1a, KU812, NC1-H1417, U-937 and WER1-RB-1 increased the production of IFN-γ in the HPP cells after 4 h of co-existence in the medium exceeds the production of IFN-γ by cells of umbilical cord blood, which grow together with the indicated cell lines during the same time period. Cm. Fig.10A. After 48 h of co-existence in the medium increased the production of IFN-γ in cells HPP superior to the products of IFN-γ in cells of umbilical cord blood for all lines of tumor cells. Cm. Fig.10V. From all lines of tumor cells cells C induce the greatest increase in production of IFN-γ in cells HPP as after 24 h and after 48 h Similar results are observed for TNF-α and GM-CSF.

The cell cycle analysis shows that the percentage of KG-1a in S-phase decreased by 30% when they are together is grown in a nutrient medium with HPP, compared with separately grown cells KG-1a. Further experiments on co-cultivation, which is carried out with the use of various enriched fractions HPP, show that antileykemichesky activity HPP largely due to the high concentration of unique immature natural killer cells, which are characterized by high level of expression of CD56+and the lack of expression of CD16.

6.6. Example 6: Inhibition of proliferation of tumor cells in vivo cells placental perfusion solution person

6.6.1. Material and methods

This example shows the efficiency of placental perfusion solution inin vivoagainst tumor the notches when using the model of xenograft tumors in mice NOD/SCID.

Growing cells KG-1 in the nutrient medium. Cells KG-1 stand in an environment of Dulbecco modification Iscove, supplemented with 20% serum, fetal cow (nutrient medium), at 37°C in a gas mixture of 95% air/5% CO2and a relative humidity of 100%. The medium in the culture change every two days and cells subcultured weekly. Cells KG-1 increase in the form of suspensions. Therefore, to change the environment or the preservation of cells collect the cell suspension in the centrifuge tubes and centrifuged with a speed of 2000 rpm for 10 min on the rotor SORVALL® HERAEUS® (part # 75006434). The supernatant discarded and the corresponding number of clot cells again suspended in a nutrient medium for further cultivation.

Preparation of cells KG-1 for implantation.For implantation of the cells into mice, the cells are harvested by centrifugation as described above. Collect clumps of cells and re-suspended them in phosphate buffered saline solution. To determine the number of cells that will be implanted mice, they calculate an aliquot of the cell suspension using hemacytometer. To exclude non-viable cells in suspension using dye tripney blue.

Preparation of HPP cells for implantation. For storage and thawing HPP samples get frozen in a dry transport con is anare in good condition. Samples stored in a dry shipping container to 7 February 2007, when they thaw. The day spend thawing, samples HPP extract from cryohalolentis (on stage) and placed in plastic bags with open top. The package is then placed in a water bath with a temperature of 37°C with gentle stirring to withstand almost complete thawing (in the package remains small frozen piece). Then the packages are removed from the bath, extract samples from packages with open top and carefully turn up until the samples are completely thawed. The samples are then placed in a fume hood laminar flow and the outer surface of the bag with blood sterilized by spraying with 70% ethanol. The bags of blood cut using sterile scissors, the cells are transferred into sterile conical tubes with a capacity of 50 ml (1 tube for each sample HPP; 2 tubes for each sample UCB) using a sterile pipette. Then in each tube slowly add 10 ml buffer solution for thawing (2.5% human albumin, 5% dextran 40) and the contents of each tube gently mixed (for a 2.2 and 2.9 min). Each bag of blood then rinse with 10 ml of buffer solution for thawing, which is then slowly poured into conical tubes with a capacity of 50 ml (for 0,7-1,3 min).

P is after thawing, each sample before centrifugation stored on melting ice. All the tubes centrifuged for 10 min (440×g at 10°C), the supernatant removed using a sterile pipette, and blood clots carefully break, shaking the tube. An aliquot of 1 ml of medium (PBS + 1% serum fetal cow) is added to one of the test tubes and the contents of the tube mixed gently rotating the tube in a circular motion. Using a pipette, capacity 2 ml of the contents is transferred to the second tube, then in a third test tube and then into the fourth tube. Drained the tube is washed with 0.2 ml of buffer solution for dilution.

For counting cells in an aliquot 25 ál is transferred into a conical tube with a capacity of 15 ml, which contains 975 ál of media, placed in ice. The erythrocytes are then subjected to lysis by adding 4 ml of cold reagent for lysis containing ammonium chloride, and incubated on ice for 10 min After incubation in each tube add 5 ml of cold PBS, and then the tubes centrifuged (10 min, 400×g, 10°C). After lysis of erythrocytes shall count cells using hemocytometer using dye tripney blue to establish the viability of the cells. The results of the calculations correct for dilution, and then divided by the ratio of lysis (0,46) to estimate the number of cells present before lysis of erythrocytes.

So is prigotovit dosage HPP, after counting cells HPP dilute to a concentration of 1×108cells/ml by adding the media. Then before napravleniya syringes cells HPP store on ice. The elapsed time between defrosting of the first sample and the completion of the preparation of the dose is less than 3 hours

Before filling the syringe aliquot 50 ál dosing substances put aside to check after a dose by counting, as described above. After a dose of remaining substance for dispensing examined to confirm dose.

The plan of the experiment. Day 1 twenty-four male mice NOD/SCID (Jackson Laboratories) subcutaneously implanted in the lateral region of 5 million viable cells KG-1. Mice are divided so that four to five mice were microisolation system cells with a bed of sawdust. Them in abundance provide sterilized food for rodents and water. Mice twice a week and thoroughly inspected for controlling tumor growth. The first tumor that can be measured, detected at Day 25. Then once a week, record the weight and tumor size was measured twice weekly with calipers. At Day 52 after implantation, animals are randomly assigned to three separate groups, while the average volume of tumors is approximately 300-350 mm3. Cm. below is table 13. The first group consists of four control mice, the mean tumor volume which is equal to 312 mm3. Two of these mice implanted intravenously (IV), and the other two are implanted intratumorally (IT) 200 ál and 50 ál of a solution of the carrier, respectively. The second group, the average tumor size which is equal to 345 mm3, consists of four mice, treated intravenously implanted with 200 ál of HPP cells per mouse (2×107cells). The last group implanted intratumorally 50 μl of HPP cells per mouse, also consists of four mice, the mean tumor volume which is 332 mm3.

Table 13
Experimental groups for ain vivoexperiments on the inhibition
The number of animalThe group, which is processed by HPPTumor volume at the day of implantation HPP
Group 1 (control)
IV 1457
IT 2429
IT 3214
IV 4147
Average:312
Group 2 (IV implantation of cells)
1466
2209
3217
4487
Average:345
Group 3 (IT implantation of cells)
1491
2256

3296
4285
Average:332
IV - implantation 200 µl; IT - implantation 50 ál

On Day 66, 14 days after implantation of cells HPP, research stop due to the fact that tumors reach too large.

6.6.2. Results

The volume of tumors (TV) measured until Day 66 (14 days after implantation of cells HPP) when the TV control group averages 2921 mm3. The group, whose introduction was performed intravenously, at the end of the study had the largest TV that is equal to the average 2076 mm3and the group, which the administration conducted intratumoral, had the largest TV that is equal to the average 2705 mm3. As for the % increase in TV after treatment, the group IT shows a moderate 20% inhibition, whereas group IV shows more than 35% inhibition of tumor growth, compared with the control group. Inhibition in the group IT can be demonstrated. Cm. Fig.11.

Equivalents:

Scope of the present invention should not be restricted described in this description of the specific variants of implementation of the image is to be placed. Indeed, various modifications of the present invention, in addition to the above in the present description, will be obvious from the preceding description and accompanying figures. It should be understood that such modifications are included in the scope of the attached formula of the present invention.

Everything described in this description reference entirety and for all purposes included in the present application by reference as if each individual publication, patent or patent application is specifically and individually were specified for inclusion in this application by reference in its entirety for all purposes. The citation of any publication is for its disclosure prior to the date of registration and it should not be seen as a recognition that the present invention could not anticipate such a publication thanks to the earlier invention.

1. The use of cells placental perfusion solution person in getting medication to suppress the proliferation of tumor cells in an individual having tumor cells, where the cells of the placental perfusion solution represents a set of nucleated cells from placental perfusion solution.

2. Application under item 1, where the cells of the placental perfusion solution containing at least 50% of the cells CD56+the placenta.

3. Application under item 2, where cells CD56 +advanced CD16-.

4. Application under item 1, where the cells of the placental perfusion solution is injected in a ratio of from 5 to 10 cells perfusion solution per tumor cell.

5. The use of natural killer cells CD56+, CD16-obtained from the placenta, in getting medication to suppress the proliferation of tumor cells in an individual having tumor cells.

6. Application under item 5, where these natural killer cells obtained from the placenta, Express one or more molecules of the microRNA hsa-miR-100, hsa-miR-127, hsa-miR-211, hsa-miR-302c, hsa-miR-326, hsa-miR-337, hsa-miR-497, hsa-miR-512-3p, hsa-miR-515-5p, hsa-miR-517b, hsa-miR-517c, hsa-miR-518a, hsa-miR-518e, hsa-miR-519d, hsa-miR-520g, hsa-miR-520h, hsa-miR-564, hsa-miR-566, hsa-miR-618, and/or hsa-miR-99a with a higher level than the natural killer cells in peripheral blood.

7. Application under item 5, where these natural killer cells obtained from the placenta, in contact with a number of immunomodulatory compounds and for a time sufficient to ensure that these natural killer cells obtained from the placenta, expressed detective more granzyme B, than an equivalent number of natural killer cells obtained from the placenta, which had no contact with specified immunomodulatory compound.

8. Application under item 5, where natural killer cells, obtained the from the placenta, contact with a number of immunomodulatory compounds and for a time sufficient to ensure that these natural killer cells obtained from the placenta showed detektiruya greater cytotoxicity against these tumor cells than an equivalent number of natural killer cells obtained from the placenta, which had no contact with specified immunomodulatory compound.

9. Application under item 7 or 8, wherein the immunomodulatory compound is lenalidomide or pomalidomide.

10. Application under item 7 or 8, where these natural killer cells obtained from the placenta, Express one or more of the WAH, CCL5, CCR5, CSF2, FAS, GUSB, IL2RA or TNFRSF18 with a higher level than an equivalent number of natural killer cells obtained from the placenta, which had no contact with specified immunomodulatory compound.

11. Application under item 7 or 8, where these natural killer cells obtained from the placenta, Express one or more of the ASTV, WAH, CCL2, CCL3, CCL5, CCR5, CSF1, CSF2, ECE1, FAS, GNLY, GUSB, GZMB, IL1A, IL2RA, IL8, IL10, LTA, PRF1, PTGS2, SKI and TBX21 with a higher level than an equivalent number of natural killer cells obtained from the placenta, which had no contact with specified immunomodulatory compound.

12. The combined organic cells-killer is in in getting medicines to suppress the proliferation of tumor cells in the individual, having tumor cells, where these combined natural killer cells include natural killer cells isolated from placental perfusion solution, and natural killer cells isolated from umbilical cord blood, and where the umbilical cord blood is separated from the placenta, from which the specified placental perfusion solution.

13. Use PP.1, 5 or 12, where tumor cells are cancer cells in the blood.

14. Use PP.1, 5 or 12, where the tumor cells are cells of a solid tumor.

15. Use PP.1, 5 or 12, where the tumor cells are cells of primary carcinoma of the epithelial duct cells, leukemia cells, acute T-cell leukemia cells, chronic myeloid lymphoma (CML) cells, acute myelogenous leukemia cells, chronic myelogenous leukemia (CML) cells, lung carcinoma cells, adenocarcinoma of the colon, cells histiocytomas lymphoma cells, multiple myeloma cells, colorectal carcinoma cells, colorectal adenocarcinoma or retinoblastoma cells.

16. Application under item 12, where these combined natural killer cells include:
the greater number of natural killer cells CD3-CD56+CD16-than an equivalent number of natural killer cells from peripheral blood;
fewer natural glue is OK-killers CD3 -CD56+CD16+than an equivalent number of natural killer cells from peripheral blood;
the greater number of natural killer cells CD3-CD56+KIR2DL2/L3+than an equivalent number of natural killer cells from peripheral blood;
fewer natural killer cells CD3-CD56+NKp46+than an equivalent number of natural killer cells from peripheral blood;
the greater number of natural killer cells CD3-CD56+NKp30+than an equivalent number of natural killer cells from peripheral blood;
the greater number of natural killer cells CD3-CD56+2B4+than an equivalent number of natural killer cells from peripheral blood; or
the greater number of natural killer cells CD3-CD56+CD94+than an equivalent number of natural killer cells from peripheral blood.

17. Application under item 16, where these natural killer cells are not grown in a nutrient medium.

18. Application under item 12, where these combined natural killer cells include:
fewer natural killer cells CD3-CD56+KIR2DL2/L3+than an equivalent number of natural killer cells from peripheral blood;
the greater number of natural killer cells CD3-CD56+NKp46+ than an equivalent number of natural killer cells from peripheral blood;
the greater number of natural killer cells CD3-CD56+NKp44+than an equivalent number of natural killer cells from peripheral blood;
the greater number of natural killer cells CD3-CD56+NKp30+than an equivalent number of natural killer cells from peripheral blood.

19. Application under item 18, where these natural killer cells are cultivated in a nutrient medium.

20. Application under item 19, where these natural killer cells are cultivated in a nutrient medium for approximately 21 days.

21. The method of suppressing the proliferation of tumor cells in vitro, comprising contacting tumor cells with cells of placental perfusion solution human cells placental perfusion solution represents a set of nucleated cells from placental perfusion solution.

22. The method according to p. 21, where the cells of the placental perfusion solution containing at least 50% of the cells CD56+the placenta.

23. The method according to p. 21, where the cells of the placental perfusion solution and the tumor cells are in contact in a ratio of from 5 to 10 cells perfusion solution per tumor cell.

24. The method of suppressing the proliferation of tumor cells in vitro, comprising contacting tumor cells with a variety of natural cell-kill the ditch, obtained from the placenta CD56+, CD16-.

25. The method according to p. 24, where these natural killer cells obtained from the placenta, Express one or more molecules of the microRNA hsa-miR-100, hsa-miR-127, hsa-miR-211, hsa-miR-302c, hsa-miR-326, hsa-miR-337, hsa-raiR-497, hsa-miR-512-3p, hsa-miR-515-5p, hsa-raiR-517b, hsa-miR-517c, hsa-miR-518a, hsa-miR-518e, hsa-miR-519d, hsa-miR-520g, hsa-miR-520h, hsa-miR-564, hsa-miR-566, hsa-miR-618, and/or hsa-miR-99a with detektivami higher level than the natural killer cells in peripheral blood.

26. The method according to p. 24, where these natural killer cells obtained from the placenta, in contact with a number of immunomodulatory compounds and for a time sufficient to ensure that these natural killer cells obtained from the placenta, expressed detective more granzyme B, than an equivalent number of natural killer cells obtained from the placenta, which had no contact with specified immunomodulatory compound.

27. The method according to p. 24, where natural killer cells obtained from the placenta, in contact with a number of immunomodulatory compounds and for a time sufficient to ensure that these natural killer cells obtained from the placenta showed detektiruya greater cytotoxicity against these tumor cells than an equivalent number of natural cell-kill the s, obtained from the placenta, which had no contact with specified immunomodulatory compound.

28. The method according to p. 26 or 27, where the immunomodulatory compound is lenalidomide or pomalidomide.

29. The method according to p. 26 or 27, where these natural killer cells obtained from the placenta, Express one or more of BAX, CCL5, CCR5, CSF2, FAS, GUSB, IL2RA or TNFRSF18 with a higher level than an equivalent number of natural killer cells obtained from the placenta, which had no contact with specified immunomodulatory compound.

30. The method according to p. 26 or 27, where these natural killer cells obtained from the placenta, Express one or more of ACTB, BAX, CCL2, CCL3, CCL5, CCR5, CSF1, CSF2, ECE1, FAS, GNLY, GUSB, GZMB, IL1A, IL2RA, IL8, IL10, LTA, PRF1, PTGS2, SKI and TBX21 with a higher level than an equivalent number of natural killer cells obtained from the placenta, which had no contact with specified immunomodulatory compound.

31. The method of suppressing the proliferation of tumor cells in vitro, comprising contacting tumor cells with combined natural killer cells, where these combined natural killer cells include natural killer cells isolated from placental perfusion solution, and natural killer cells isolated from umbilical cord blood, and where the umbilical cord blood is extracted from platen is s, from which the specified placental perfusion solution.

32. The method according to PP.21, 24 or 31, where tumor cells are cancer cells in the blood.

33. The method according to PP.21, 24 or 31, where the tumor cells are cells of a solid tumor.

34. The method according to PP.21, 24 or 31, where the tumor cells are cells of primary carcinoma of the epithelial duct cells, leukemia cells, acute T-cell leukemia cells, chronic myeloid lymphoma (CML) cells, acute myelogenous leukemia cells, chronic myelogenous leukemia (CML) cells, lung carcinoma cells, adenocarcinoma of the colon, cells histiocytomas lymphoma cells, multiple myeloma cells, colorectal carcinoma cells, colorectal adenocarcinoma or retinoblastoma cells.

35. The method according to p. 31, where these combined natural killer cells include:
the greater number of natural killer cells CD3-CD56+CD16-than an equivalent number of natural killer cells from peripheral blood;
fewer natural killer cells CD3-CD56+CD16+than an equivalent number of natural killer cells from peripheral blood;
the greater number of natural killer cells CD3-CD56+KIR2DL2/L3+than an equivalent number of natural killer cells from peripheral blood;
Myung is the neck of the number of natural killer cells CD3 -CD56+NKp46+than an equivalent number of natural killer cells from peripheral blood;
the greater number of natural killer cells CD3-CD56+NKp30+than an equivalent number of natural killer cells from peripheral blood;
the greater number of natural killer cells CD3-CD56+2B4+than an equivalent number of natural killer cells from peripheral blood; or
the greater number of natural killer cells CD3-CD56+CD94+than an equivalent number of natural killer cells from peripheral blood.

36. The method according to p. 35, where these natural killer cells are not grown in a nutrient medium.

37. The method according to p. 31, where these combined natural killer cells include:
fewer natural killer cells CD3-CD56+KIR2DL2/L3+than an equivalent number of natural killer cells from peripheral blood;
the greater number of natural killer cells CD3-CD56+NKp46+than an equivalent number of natural killer cells from peripheral blood;
the greater number of natural killer cells CD3-CD56+NKp44+than an equivalent number of natural killer cells from peripheral blood;
the greater number of natural killer cells CD3-CD56+NKp30+than equivalent is lentsoe the number of natural killer cells from peripheral blood.

38. The method according to p. 37, where these natural killer cells are cultivated in a nutrient medium.

39. The method according to p. 38, where these natural killer cells are cultivated in a nutrient medium for approximately 21 days.

40. Composition for use in inhibiting proliferation of tumor cells containing selected natural killer cells CD56+, CD16-where these natural killer cells isolated from placental perfusion solution and where these natural killer cells constitute at least 50% of the cells in the composition.



 

Same patents:

FIELD: medicine, pharmaceutics.

SUBSTANCE: group of inventions refers to biotechnology and oncology. A method provides: a) isolating postnatal tissue-specific pluripotent autologous stem cells (ASCs) and/or autologous progenitor cells (APCs) for the following proteome and complete transcriptomic analysis; b) isolating ASCs and/or APCs and/or pluripotent allogenic HLA-haploidentical stem cells (HLA-SCs) for remodelling of their proteome profile; c) isolating cancerous stem cells from patient's tumour; d) carrying out ASC and/or APC and CSC proteome analysis; e) carrying out ASC and/or APC and CSC complete transcriptomic analysis; f) recognising a protein complement each of which is found in the proteome profiles both of ASCs and/or APCs, and of CSCs; g) analysing the recognising protein complement for identifying intracellular signalling pathways in CSCs not subject to the neoplastic transformation as a result of carcinogenesis, and recognising target proteins defined as membrane acceptors of identified signalling pathways; h) analysing the complete transcriptomic profile of the CSC gene expression and confirming the preservation and functional significance of the structural components of the identified signalling pathways in CSCs; i) recognising ligand proteins able to activate the target proteins; j) carrying out a comparative analysis of the ASC and/or APC complete transcriptomic profiles to the transcriptomic profiles contained in the known data bases of transcriptomes for recognising perturbagens able to modify the gene expression profile of ASCs and/or APCs and/or HLA-SCs isolated for remodelling their proteome profile in the line of secreting the pre-recognised ligand proteins; k) remodelling the ASC and/or APC and/or HLA-SC proteome profile with perturbagens to produce the modified transcriptome profile of various cell systems able to have a regulatory influence on patient's CSCs.

EFFECT: preparation produced according to the method includes all the individual peculiarities of the patient's genome and proteome modifications, and has the regulatory influence on patient's cancerous stem cells (CSCs) and malignant cells.

8 cl, 4 dwg, 11 tbl, 4 ex

FIELD: biotechnologies.

SUBSTANCE: population of mononuclear cells or non-germ stem cells, which is saturated with cells of monocytic lineage containing promonocytes, is used for treatment of ischemia with a patient.

EFFECT: invention allows effective treatment of ischemia with a patient by injection of the above population of therapeutical cells to ischemic tissue of the patient.

13 cl, 8 dwg, 5 tbl, 3 ex

FIELD: medicine.

SUBSTANCE: invention relates to the field of biotechnology and cell technology. The claimed invention is aimed at the creation of pluripotent, multipotent and/or self-renewing cells, which are able to start differentiating in a culture into various types of cells and are capable of further differentiation in vivo. The claimed invention is also aimed at the creation of populations of the required differentiating cells, which can be transplanted to patients, genetic modification of endogenic cells and treatment of patients, suffering from diseases, intensity of which can be reduced by means of the said methods.

EFFECT: invention also claims methods of prevention, treatment or retardation of a disease, associated with an infection of immunodeficiency virus.

17 cl, 1 dwg, 13 ex

FIELD: medicine.

SUBSTANCE: method provides placing a sperm cell drop and a culture medium drop in the Petri dish at a distance from each other of no more than 5 cm, coupling the drops with a viscous medium strip having a viscosity of 1-4 Pa·s, incubating the dish with its content for 30-90 min in the environment simulating natural environment of the female cervical canal. Before placing into the Petri dish, the culture medium and the viscous medium is incubated until pH value of 7.2-7.6 is achieved.

EFFECT: method enables higher quality of sperm cells selection possessing the highest fertility ability for extracorporeal fertilisation.

11 cl, 7 dwg, 2 tbl

FIELD: medicine.

SUBSTANCE: invention refers to medicine, biotechnology and cell technologies. A method for differentiating pluripotent stem cells presenting a human cell line in cells expressing markers specific for a formed endoderm line involves preparing the pluripotent stem cells in a medium differing by the fact that it is free from activin A and contains GDF-8 for the period of time adequate to differentiate the pluripotent stem cells in the cells expressing the markers specific for a formed endoderm line.

EFFECT: invention can be used in medicine for transplantation applications.

14 cl, 19 tbl, 27 dwg, 24 ex

FIELD: biotechnologies.

SUBSTANCE: invention proposes oligopeptide versions extracted from protein RAB6KIFL (KIFL20A), which can induce cytotoxic T lymphocytes (CTL) consisting of a complex with molecule HLA-A*0201. Besides, the following has been considered: a pharmaceutical composition and an elimination method of cells expressing RAB6KIFL and HLA-A*0201, an exosome, an extracted antigen-presenting cell and a method for its induction, a pharmaceutical composition and a method of CTL induction, use of active ingredients as per this invention to obtain a pharmaceutical composition for cancer treatment, as well as a polynucleotide coding the oligopeptide as per this invention.

EFFECT: invention can be further used in therapy of diseases associated with RAB6KIFL.

13 cl, 6 dwg, 1 tbl

Brewage method // 2531522

FIELD: food industry.

SUBSTANCE: invention relates to the field of food industry and represents a brewage method involving thermostable protease addition to the wort after the latter filtration but before cooking; protease thermostability means that such protease activity accounts for at least 70% of its activity measured in the following way: protease is diluted till concentration equal to 1 mg/ml in an analytic buffer (containing 100 mmol of succinic acid, 100 mmol of HEPES, 100 mmol of CHES, 100 mmol of CABS, 1 mmol of CaCl2, 150 mmol of KCl, 0.01% Triton X-100) with pH conditioned to 5.5 with the help of NaOH; the protease is pre-incubated i) in ice and ii) for 10 minutes at a temperature of 70°C; the substrate in relation whereto protease displays activity is suspended in 0.01% Triton X-100: for reaction beginning protease is added in an amount of 20 mcl into a test tube and incubated in an Eppendorf thermomixer at 70°C, 1400 rpm during 15 minutes; the reaction is stopped by way of the test tubes placement into ice; the samples are centrifuged in a cold condition at 14000 g during 3 minutes; the supernatant optic density OD590 is measured; the obtained OD590 value of samples without protease is subtracted from the obtained OD590 value of samples treated with protease; protease thermal stability is determined by way of calculation of protease percentage activity in the samples pre-incubated at a temperature of 70°C relative to protease activity in the samples incubated in ice as 100%-activity.

EFFECT: invention allows to enhance colloidal stability of wort and beer as well as preserve the level of total nitrogen in wort and beer due to protease addition to filtered wort.

16 cl, 1 dwg, 17 tbl, 7 ex

FIELD: medicine.

SUBSTANCE: invention refers to cell biology, cell transplantology and tissue engineering. A method for increasing the angiogenic activity of stromal cells of the fatty tissue in tissues and organs involves recovering the stromal cells of the fatty tissue, culturing the recovered cells in the presence of tumour necrosis factor-alpha in amounts of 5 or 100 ng/ml for 24-72 hours, and transplanting into the tissues or organs.

EFFECT: invention can be used for repairing the injured tissues and arresting an ischemia-related developing pathology.

3 cl, 11 dwg

FIELD: medicine, pharmaceutics.

SUBSTANCE: inventions deal with a membrane, used as a substrate for growing cells of retinal pigment epithelium, its application for supporting cells and a method of inoculating the cells on such a membrane. The characterised membrane is non-biodegradable and porous, covered from at least one side with a glycoprotein-containing coating, with pores with a diameter approximately from 0.2 mcm to 0.5 mcm, with a density of membrane pores constituting approximately from 1×107 to 3×108 pores per 1 cm2, and hydraulic conductivity of the membrane higher than 50×10-10 m sec-1 Pa-1,and having the maximal thickness of 11 mm.

EFFECT: claimed inventions make it possible to obtain a transplant for treatment of age-related macular degeneration.

19 cl, 4 dwg, 6 ex, 3 tbl

FIELD: medicine.

SUBSTANCE: invention relates to biotechnology, cell technologies and tissue surgery. A method for preparing a smooth muscle cell culture consists in cutting a blood vessel fragment, grinding it to a piece size of no more than 2 mm in any dimension, and incubating the pieces in a culture flask having its bottom preliminarily scratched and containing a culture medium containing 10% embyo foetal serum for at least ten days, but no more than 24 days at 37°C in the CO2 incubator environment; the method differs by the fact that the above blood vessel fragment is an ascending thoracic aorta fragment cut during the coronary artery bypass surgery; before the incubation, the above pieces of the ascending thoracic aorta fragment are kept in the culture medium containing 0.1% collagenase for at least 30 minutes, but no more than 60 minutes at 37°C and then washed in the cell culture medium.

EFFECT: invention enables preparing the cells of the aortic tissue directly from the patient's vital tissues for transplantology applications.

3 dwg

FIELD: chemistry.

SUBSTANCE: invention relates to field of organic chemistry, namely to heterocyclic compound of formula (I) or its racemate, enantiomer, diastereoisomer and their mixture, as well as to their pharmaceutically acceptable salt, where A is selected from the group, consisting of carbon atom or nitrogen atom; when A represents carbon atom, R1 represents C1-C6-alkoxyl; R2 represents cyano; when A represents nitrogen atom, R1 hydrogen atom or C1-C6-alkoxyl; where said C1-C6-alkoxyl is optionally additionally substituted with one C1-C6-alkoxyl group; R2 is absent; R3 represents radical, which has the formula given below: or , where D represents phenyl, where phenyl is optionally additionally substituted with one or two halogen atoms; T represents -O(CH2)r-; L represents pyridyl; R4 and R5 each represents hydrogen atom; R6 and R7 each is independently selected from hydrogen atom or hydroxyl; R8 represents hydrogen atom; R9 represents hydrogen atom or C1-C6-alkyl; r equals 1 and n equals 2 or 3. Invention also relates to intermediate compound of formula (IA), method of obtaining compound of formulae (I) and (IA), pharmaceutical composition based on formula (I) compound and method of its obtaining and to application of formula (I) compound.

EFFECT: novel heterocyclic compounds, inhibiting activity with respect to receptor tyrosine kinases EGFR or receptor tyrosine kinases HER-2 are obtained.

18 cl, 12 ex, 4 tbl

FIELD: medicine, pharmaceutics.

SUBSTANCE: present invention refers to immunology. What is presented is a polypeptide containing two binding fragments presented by antibodies; the first of them binds to CD3e(epsilon) chain epitope of a human or a primate, other than a chimpanzee, particularly Callithrix jacchus, Saguinus oedipus and Saimiri sciureus; the second one - to EGFR, Her2/neu or IgE of a human or a primate, other than a chimpanzee, with the above CD3e epitope containing the amino acid sequence Gln-Asp-Gly-Asn-Glu. There are also disclosed a coding sequence of the nucleic acid, a vector, a host cell and a method for preparing the above peptide, as well as a pharmaceutical composition and using the polypeptide in preventing, treating or relieving a proliferative disease, a malignant disease or an immunological disorder.

EFFECT: invention provides the clinical improvement of T-cell redistribution and the enhanced safety profile.

17 cl, 8 tbl, 26 dwg, 26 ex

FIELD: biotechnology.

SUBSTANCE: invention relates to a novel compound, namely 2,2a,2a',3,5a,9b-hexahydrofluorene[9,1-bc]furan-8-ol of the formula 1 .

EFFECT: enhancement of antitumor activity.

2 dwg, 5 tbl, 1 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention relates to novel fluorinated 1,4-naphthoquinone derivatives of general formula , where 1 R=C6H5, X=F; 2 R=2, 5-F2C6H3, X=F; 3 R=CH3, X=F; 4 R=C6H5, X, , possessing cytotoxic activity with respect to cancer cells, which can be applied in medicine.

EFFECT: claimed are novel compounds with anti-cancer activity for therapy of malignant neoplasms.

1 dwg, 3 tbl, 5 ex

FIELD: medicine.

SUBSTANCE: invention relates to the field of biotechnology and cell technology. The claimed invention is aimed at the creation of pluripotent, multipotent and/or self-renewing cells, which are able to start differentiating in a culture into various types of cells and are capable of further differentiation in vivo. The claimed invention is also aimed at the creation of populations of the required differentiating cells, which can be transplanted to patients, genetic modification of endogenic cells and treatment of patients, suffering from diseases, intensity of which can be reduced by means of the said methods.

EFFECT: invention also claims methods of prevention, treatment or retardation of a disease, associated with an infection of immunodeficiency virus.

17 cl, 1 dwg, 13 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to compounds of formula

wherein A1 represents N or C (A2); A2 represents H, F, Cl or CN; B1 represents H, OR1, SO2R1, NHR1, NHC(O)R1, F or Cl; D1 and E1 represents H or Cl; Y1 represents H, CN, NO2, F, Cl, Br, CF3, R17, OR17, SO2R17 or C(O)NH2; or Y1 and B1 together with atoms to which they are attached, represent 5- or 6-merous heteroarene having 2-3 nitrogen atoms, wherein heteroarene rings are unsubstituted or substituted by (O); G1 represents H; Z1 represents uncondensed phenylene substituted by OR41; R41 represents 6-merous heteroaryl having 1 N atom, wherein heteroaryl is condensed with R43A, R43A represents 5-merous heteroarene having 1 N atom; Z2 represents monocyclic 6-merous heterocycloalkylene having 1-2 N atoms and 0 double bonds; Z1A and Z2A are both absent; L1 represents -CH2-; Z3 represents R38 or R40; R38 represents uncondensed phenyl; R40 represents cycloalkyl, wherein cycloalkyl represents a monocyclic ring system having 3 to10 C atoms and 0 double bonds, cycloalkenyl, wherein cycloalkenyl represents monocyclic 6-merous ring having 1 heteroatom specified in a group consisting of O and N, and 1 double bond, wherein cycloalkenyl is uncondensed or condensed with R40A; R40A represents cycloalkane, wherein cycloalkane represents a monocyclic ring having 3-10 C atoms and 0 double bonds, or heterocycloalkane, wherein heterocycloalkane represents monocyclic 6-merous ring having 1 N atom and 0 double bonds (the rest substitutes are those as specified in cl. 1 of the patent claim). The invention also refers to compounds of formula

and a pharmaceutical composition containing an effective amount of the compound of formula (I) or (II) or its pharmaceutically acceptable salt.

EFFECT: compounds of formula (I) or (II) inhibiting the activity of anti-apoptotic Bcl-2 proteins.

6 cl, 5 tbl, 378 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention relates to novel compounds of general formula [1] or their pharmaceutically acceptable salts, which possess properties of an inhibitor of the JAK2 thyrokinase activity. In general formula radicals are selected from group (I) or (II). In group (I) X represents CH or N; R1 represents a halogen atom and R2 represents H, a halogen atom, CN, or is selected from the groups of formulas

,

or a group -ORP or 5-6-membered heteroaryl, containing 1-4 nitrogen atoms and optionally additionally containing an oxygen or sulphur atom or containing an oxygen atom as a heteroatom, optionally substituted; or (II) X represents -CRA; and RA represents a group of formula , RB represents (a) amino, optionally substituted with one or two groups, selected from the group, consisting of C1-6alkyl, C3-6cycloalkyl, (C3-6cycloalkyl)C1-6alkyl and C1-3alcoxyC1-3alkyl, (b) C1-3alcoxy, (c) hydroxy or (d) a 5-6-membered saturated cyclic amino group, which additionally can contain a heteroatom, selected from an oxygen atom; R1 represents a halogen atom and R2 represents H; R3 -R5 have values given above. Other values of the radicals are given in the invention formula.

EFFECT: compounds can be applied for the prevention or treatment of cancer, for instance hematologic cancer disease or a solid form of cancer, inflammatory disorder, for instance, rheumatoid arthritis, inflammatory intestinal disease, osteoporosis or multiple sclerosis and angiopathy, for instance, pulmonary hypertension, arteriosclerosis, aneurism or varicose veins.

14 cl, 19 tbl, 234 ex

FIELD: medicine, pharmaceitics.

SUBSTANCE: invention relates to particular derivatives of N-(phenylsulphonyl)benzamide, given in i.1 of the invention formula. The invention also relates to a pharmaceutical composition, possessing an inhibiting activity with respect to anti-apoptotic proteins Bcl-2, containing an effective quantity of one of the said compounds or a therapeutically acceptable salt of such a compound.

EFFECT: N-(phenylsulphonyl)benzamide derivatives as inhibitors of the anti-apoptotic proteins Bcl-2.

2 cl, 2 tbl, 458 ex

FIELD: chemistry.

SUBSTANCE: invention relates to the field of cosmetology. Described is a stable and safe antioxidant composition, which can be applied daily. In particular, described is the antioxidant composition, which contains one or more compounds, selected from the group, which consists of D-aspartic acid, its derivatives and/or its salts. The composition can be applied with the purpose of suppressing and/or relief of a skin condition. Conditions of skin can include, but are not limited by them, small wrinkles, rough skin, dry skin, skin cancer, skin allergy, skin inflammation, and light-sensitive dermatosis. The composition can be applied as a medication for the external application on the skin.

EFFECT: invention ensures an increase of the antioxidant effect of the composition.

4 cl, 5 dwg, 31 ex

FIELD: chemistry.

SUBSTANCE: claimed is method of obtaining photosensibiliser, which consists in the following: 3-pyridylcarboxaldehide is condensed with pyrrole in mixture propionic acid-propionic anhydride with their ratio 3-4:1-2 in boiling for 80-100 min. product of condensation is reduced with p-toluenesulfonylhydrazide in pyridine medium in presence of potassium carbonate with its 30-35-fold excess and 5-10% quinoline at temperature 85-95°C for 4-5 hours. After that, obtained product is N-methylated in dimethylformamide in boiling for 50-65 min, precipitated with benzene, filtered and dried. Also claimed is photosensibiliser, obtained in accordance with claimed method, which contains 5,10,15,20-tetrakis(N-methyl-3'-pyridyl)chlorine in quantity 15-25% and 5,10,15,20-tetrakis(N-methyl-3'-pyrydyl)bacteriochlorine in quantity 75-85%.

EFFECT: increase of target product output, reduction of tumour growth rate and dissemination, prevention of tumout tissue necrotisation, complete and uniform saturation of tumour tissue with medication.

3 cl, 1 dwg, 1 tbl, 2 ex

FIELD: veterinary medicine.

SUBSTANCE: method comprises administering an agent containing gentamicin, gamavit, sodium benzoate, potassium sorbate and propylene glycol. The agent is used in intrauterine mode 2 times a day for 7-10 days at a dose of 5-10 ml to cat and 10-20 ml to dog.

EFFECT: method comprises high therapeutic efficacy in endometritis of cats and dogs.

1 ex

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